BIOLOGY  LIBRAflt 


A   MANUAL 

OF 


CLINICAL    CHEMISTRY,    MICROSCOPY, 
AND  BACTERIOLOGY 


A    MANUAL 

OF 

CLINICAL  CHEMISTRY,  MICROSCOPY, 
AND  BACTERIOLOGY 

BY 
DR.  M.  KLOPSTOCK   AND   DR.  A.  KOWARSKY 

OF     BERLIN 

In  their  "Institut  fur  medizinische  Diagnostik,"  in  Berlin 


ONLY   AUTHORIZED    TRANSLATION   FROM    THE   LAST 

GERMAN   EDITION,   THOROUGHLY  REVISED 

AND    ENLARGED 


ILLUSTRATED   WITH  FORTY-THREE  TEXTUAL  FIGURES 
AND  SIXTEEN  COLORED  PLATES 


NEW   YORK 

REBMAN    COMPANY 

1123   BROADWAY 


310LOSY 
UDRARY 


COPYRIGHT,  1912,  BY 

REBMAN   COMPANY 

NEW  YORK 

All  Rights  Reserved 


PRINTED   IN   AMERICA 


PREFACE  TO  THE   GERMAN    EDITION 

THIS  book  owes  its  existence  to  the  desire  of  the 
authors  to  place  a  concise  manual  in  the  hands  of  those 
taking  part  in  the  course  in  Clinical  Chemistry,  Micro- 
scopy, and  Bacteriology  held  in  their  ' '  Institut  fur  medi- 
zinische  Diagnostic, "  in  Berlin.  It  is  in  nowise  intended 
to  replace  larger  and  more  elaborate  text-books,  but  aims 
to  present  in  concise  form  the  essential  features  of  the  sub- 
jects treated.  As  the  book  is  intended  especially  for  the 
practitioner,  we  have  assumed  that  the  reader  has  an 
elementary  chemical  and  bacteriological  education.  For 
the  same  reason  the  needs  of  daily  practice  have  been 
especially  considered  in  the  choice  of  the  methods  of 
examination.  Wherever  it  has  been  possible  the  simplest 
and  quickest  methods  have  been  chosen. 

It  would  be  a  source  of  gratification  to  us  if  this  book 
should  find  favor  in  wider  medical  circles. 

THE  AUTHORS. 


248545 


PUBLISHERS'  ANNOUNCEMENT 

HAVING  found  this  book  a  valuable  laboratory  guide, 
it  is  a  pleasure  to  comply  with  the  request  of  the 
Authors  to  publish  an  English  Translation  of  this  New 
Edition.  The  Authors  have  retained  all  those  matters 
which  by  experience  have  proved  themselves  to  be  of 
genuine,  practical  value  to  the  student  as  well  as  to  the 
medical  man  in  General  Practice.  The  pages  relating  to 
Typhoid  Fever  and  to  the  Meningococci  have  been  re- 
written with  great  care;  whilst  those  dealing  with  the 
Spirocheta  pallida  and  with  the  Wassermann  Reaction, 
especially,  are  entirely  new. 

The  book  has  been  brought  up  to  date.  The  Index 
at  the  end  of  the  volume  is  a  new  feature,  and  the  refer- 
ences to  the  pages  where  they  are  quoted  will  prove  of 
real  value  when  consulting  the  colored  plates. 

REBMAN  COMPANY. 
NEW  YORK,  1123  BROADWAY. 


CONTENTS 


CHAPTER   I 

BACTERIOLOGICAL  EXAMINATION  OF  THE  SECRETIONS 
AND  DEPOSITS  IN  THE  MOUTH  AND  PHARYNX 

PAGE 

Collection  of  Material  to  be  Examined 1 

Morphological  and  Staining  Characteristics  of  Diphtheria 

Bacilli , 2 

Cultural  Characteristics 4 

Animal  Inoculation 5 

Differential  Diagnosis 5 

Order  of  Examination 8 

Oidium  albicans  — "Soor  fungus" 10 

Angina  Vincenti  (s.  Plautii) 11 

CHAPTER   II 

BACTERIOLOGICAL  EXAMINATION  OF  NASAL   SECRETIONS 

Diphtheria  Bacilli 17 

Tubercle  Bacilli 17 

Lepra  Bacilli 18 

Diplobacillus  of  Friedlaender 18 

CHAPTER   III 

BACTERIOLOGICAL  EXAMINATION  OF  CONJUNCTIVAL 
SECRETION 

Diphtheria  Bacilli 19 

Tubercle  Bacilli 19 

Gonococci 20 

Bacilli  of  Koch  and  Weeks 20 

Diplobacillus  of  Morax  and  Axenfeld 21 

Other  Exciters  of  Conjunctivitis 21 


CONTENTS 
CHAPTER   IV 

EXAMINATION  OF  THE  SPUTUM 

PAGE 

Method  of  Obtaining  Material  for  Examination 22 

General  Characteristics 23 

Especially  Prominent  Constituents  of  the  Sputum 23 

Composition  of  Sputum 24 

Especially  Prominent  Ingredients  of  the  Sputum 26 

Microscopical  Examination 28 

Curschmanri 's  Spirals 29 

Fibrin   Coagula 30 

Dittrich's  Plugs 30 

Portions  of  the  Echinococcus 31 

Actinomyces  Granules 32 

Cellular  Elements  of  the  Sputum 33 

Elastic   Fibres 35 

Crystalline  Bodies 36 

Bacteriological  Examination  of  the  Sputum 37 

Examination  of  Stained  Smears 38 

Cultural  Examination 40 

Animal  Inoculation 40 

Detection  of  Tubercle  Bacilli 40 

Pneumococci 48 

Streptococci 49 

Staphylococci 50 

Micrococcus  tetragenus 50 

Micrococcus  catarrhalis 51 

Influenza  Bacillus 51 

Diplobacillus  of  Friedlaender 52 

Bacillus  pyocyaneus 53 

Bacillus  of  Bubonic  Plague..  53 


CHAPTER   V 

EXAMINATION  OF  THE  GASTRIC  CONTENTS 

General  Characteristics 56 

Qualitative  Chemical  Examination 58 

Reaction..  58 


CONTENTS 

PAGE 

Qualitative  Chemical  Examination : 

Free  Acids 58 

Free  Hydrochloric  Acid 58 

Lactic  Acid 61 

Volatile  Fatty  Acids 61 

Pepsin  and  Pepsinogen 62 

Renin  and  Reninogen 65 

Bile  Pigment 66 

Blood 66 

Hydrogen  Sulphide 68 

Quantitative  Chemical  Examination  of  the  Gastric  Con- 
tents   68 

Estimation  of  Total  Acidity 68 

of  Free  Hydrochloric  Acid 69 

of  Total  Hydrochloric  Acid , . . .  70 

of  Lactic  Acid 71 

Microscopical  Examination  of  the  Gastric  Contents..  72 


CHAPTER  VI 

EXAMINATION  OF  THE  FAECES 

General  Characteristics 74 

Qualitative  Chemical  Examination  of  the  Faeces 80 

Reaction 80 

Mucin 80 

Fat 81 

Blood . 81 

Biliary  Constituents 82 

Quantitative  Chemical  Examination  of  the  Faeces 84 

Estimation  of  Dry  Matter 84 

of  Total  Nitrogen 85 

of  Fat 85 

of  Carbohydrates 86 

Direct  Estimation  of  Starch  According  to  Liebermann  and 

Allihn 87 

Fermentation  Test  According  to  Schmidt 88 

Examination  of  Gall-Stones  and  Biliary  Concretions 90 

Fecal  Concretions,  Enteroliths,  and  Pancreatic  Stones  . .  92 


CONTENTS 

PAGE 

Microscopical  Examination  of  the  Faeces 94 

Food  Particles 94 

Pathological  Products  of  the  Intestinal  Wall 96 

Intestinal  Parasites  and  their  Eggs 97 

Bacteriological  Examination  of  the  Faeces 104 

Typhoid  Bacilli 104 

Characteristics  of  Typhoid  Bacilli 105 

Biological  Characteristics  of  Typhoid  Bacilli 108 

Order  of  Examination  of  the  Faeces  for  Typhoid  Bacilli 113 

Planting  of  Cultures  from  the  Faeces 113 

Examination  of  the  Plates 114 

Bacilli  in  Meat-Poisoning 117 

Dysentery  Bacilli 117 

Cholera  Vibriones 120 

Pfeiffer's  Test 122 

Detection  of  Cholera  Vibriones  in  the  Faeces 125 

Tubercle  Bacilli 126 

Staphylococci  and  Streptococci 127 

Anthrax  Bacilli 128 

Plague  Bacilli 128 


CHAPTER   VII 

EXAMINATION  OF  THE  URINE 

Collection  of  the  Urine 129 

The  Identification  of  a  Fluid  as  Urine 130 

Chemical  and  Physical  Characteristics  of  the  Urine 132 

Color : 132 

Transparency 133 

Reaction 135 

Specific  Gravity 136 

Freezing-point 137 

Quantity 140 

The  Chemical  Examination  of  the  Pathological  and  Abnor- 
mal Constituents  of  the  Urine 141 

Albumin 141 

Albumoses  and  Peptones 148 

Method  of  Freeing  the  Urine  from  Albumin 149 

Albumins  Precipitated  by  Cold  Acetic  Acid 150 


CONTENTS 

PAGE 

The  Chemical  Examination  of  Fibrin 151 

Glucose 151 

Lactose 160 

Levulose 160 

Pentose 161 

Glycuronic  Acid 162 

Acetone 163 

Diacetic  Acid 164 

/8-Oxybutyric  Acid ' 165 

Indican 165 

Urobilin 167 

Biliary  Pigments 168 

Blood  Pigments 170 

HaBmatoporphyrin 173 

Melanin 174 

Diazo  Reaction 174 

Adventitious  Constituents  of  the  Urine— Iodine,  Mer- 
cury, etc 175 

Quantitative  Chemical  Examination  of  the  Urine 180 

Estimation  of  Albumin 180 

of  Sugar 182 

of  Total  Nitrogen 187 

of  Urates 188 

of  Uric  Acid 193 

of  Chlorides 199 

of  Phosphates 199 

of  Sulphates 201 

of  Oxalic  Acid  according  to  Salkowski 202 

Examination  of  Urinary  Calculi  and  Concretions 206 

Microscopical  Examination  of  the  Urinary  Sediment 209 

Organized  Sediments 224 

Bacteriological  Examination  of  the  Urine 237 

Collection  and  Preparation  of  the  Urine  for  Examination  237 

Method  of  Examination 238 

Bacterium  coli 239 

Staphylococci  and  Streptococci 240 

Tubercle  Bacilli 240 

Typhoid  Bacilli 244 

Gonococci 244 

Proteus  vulgaris .  245 


CONTENTS 
CHAPTER  VIII 

EXAMINATION  OF  THE  URETHRAL  AND  PROSTATIC 
SECRETIONS 

Prostatic  Secretion  . . 


CHAPTER   IX 

EXAMINATION  OF  THE  BLOOD 

Determination  of  the  Specific  Gravity 250 

of  the  Freezing-point 251 

Estimation  of  Hemoglobin ]  '251 

Enumeration  of  Blood-Corpuscles '253 

Histological  Examination '  '255 

Examination  of  Fresh  Specimens 

of  Stained  Specimens 

Sketch  of  the  Mbrphology  of  the  Blood '259 

Bacteriological  Examination  of  the  Blood 263 

Examination  of  the  Blood  in  Stained  Smears. .  263 

Malaria 263 

Spirilla  of  Relapsing-  Fever 

Examination  of  the  Blood  by  Means  of  Cultural  Pro- 
cedures    geg 

Cultivation  of  Typhoid  Bacilli ............ ' .' .'  *  '  269 

of  Staphylococci  and  Streptococci  271 
Examination  of  the  Blood  by  Means  of  Animal  Inoc- 
ulation   271 

Serum  Diagnosis 272 

Macroscopic,  Quantitative  Agglutination  Test!! 

Exploratory  Agglutination  Test 272 

Widal's  Reaction  ...  '  979 

Pfeiffer'sTest .'.'.'.'.'.'.'.'.'.'.'.'  '  375 

The  Bactericidal  Test-tube  Test '  276 

Serum  Diagnosis  of  Syphilis  According to  Wasser- 

mann 27g 

Wassermann's  Reaction ]  278 


CONTENTS 
CHAPTER   X 

EXAMINATION  OF  FLUIDS   OBTAINED  BY  PUNCTURE 

PAGE 

General  Characteristics  and  Chemical  Examination 286 

Microscopical  Examination 290 

Bacteriological  Examination 291 

Collection  of  Material  for  Examination 291 

Method  of  Examination 292 

The  Most  Important  Bacteriological  Findings 295 

CHAPTER   XI 

BACTERIOLOGICAL  EXAMINATION  OF  DISEASES  OF  THE 
SKIN 

Purulent  Affections  of  the  Skin 299 

Glanders 300 

Anthrax 301 

Tetanus 302 

Bacillus  of  ulcus  molle 304 

Tuberculosis  of  the  Skin 305 

Diseases  of  the  Skin  excited  by  Hyphomycetes  (Dermato- 

mycosis) 305 

Favus 309 

Trichophytosis 311 

Tinea  sycosis 312 

' '      circinata 313 

Pityriasis  versicolor 315 

Erythrasma 316 

Spirocheta  pallida 317 

CHAPTER  xn 

THE   USUAL   METHODS    OF   BACTERIOLOGICAL   EXAMINATION, 
FORMULAE   OF  STAINS,   AND  CULTURE   MEDIA 

Examination  in  a  Hanging-Drop 327 

Examination  in  Stained  Smears 328 

Preparation  of  the  Specimens 328 


CONTENTS 

PAGE 

Examination  in  Stained  Smears : 

Staining  Methods  and  Staining  Solutions 329 

Stock  Solutions 329 

of  Tubercle  Bacilli  and  other  Acid-fast  Bacilli  330 

of  Diphtheria  Bacilli 332 

"        of  Gonococci 334 

of  Spores 334 

of  the  Capsules  of  Anthrax  Bacilli 335 

of  Flagella 336 

of  Fungi 337 

' '        of  Blood  Specimens 338 

Examination  of  Cut  Sections 339 

Universal  Staining  Methods   for   Demonstrating  Bac- 
teria in  Sections 341 

Special  Staining  Methods 342 

Cultural  Methods 344 

Preparation  of  Culture  Media 344 

The  Cultural  Methods  most  frequently  Employed 356 

Aerobic  Cultures 356 

Anaerobic  Cultures 359 

Determination  of  the  Biological  Characteristics  of  Bacteria  361 
Methods  of  Animal  Inoculation 363 

INDEX..  .  365 


LIST   OF  FIGURES   IN   THE  TEXT 

FIG.  PAGE 

1.  Oidium  Albicans — "Soor  Fungus" 10 

2.  Fibrin  Clots  from  Pneumonic  Sputum 27 

3.  Spirals  from  the  Sputum  (natural  size) 29 

4.  Spirals  from  the  Sputum  (magnified) 29 

5.  Hyaline  Membrane  of  an  Echinococcus  Cyst 31 

6.  Echinococcus  Hooks 31 

7.  Actinomyces  Granules  (low  power) 32 

8.  Actinomyces  Granules  (unstained  specimen) 33 

9.  Elastic  Fibres  from  the  Sputum 35 

10.  Ley  den  Crystals  (magnified  300  times) 36 

11.  Boas'  Fecal  Sieve 77 

12.  .  Schmidt's  Fermentation  Apparatus 89 

13.  Amoeba  Coli 98 

14.  Balantidium  Coli 98 

15.  Tsenia  Solium 99 

16.  TaBnia  Saginata 100 

17.  Scolex  of  Bothriocephalus  Latus 100 

18.  Oxyuris  Vermicularis 101 

19.  Ascaris  Lumbricoides 102 

20.  Tricocephalus  Dispar 102 

21.  Anchylostoma  Duodenale 103 

22.  Beckmaryi's  Cry oscope 138 

23.  Einhorn's  Saccharometer 157 

24.  Jolles'  Azotometer 191 

25.  Amorphous  Urates 213 

26.  Calcium  Oxalate 215 

27.  Neutral  Calcium  Phosphate ...  .216 


LIST   OF   FIGURES   IN  THE  TEXT 

PIG.  PAGE 

28.  Triple  Phosphate 218 

29.  Tyrosin— Cystin— Leucin 219 

30.  Cystin  Crystals 220 

31.  Hippuric  Acid 221 

32.  Fatty  Acid  Needles 223 

33.  Epithelial  Cells . . . .- 224 

34.  Squamous  Epithelial  Cells 226 

35.  Cylindroids 232 

36.  Urinary  Filaments 233 

37.  Substances  Found  in  the  Sediment  of  Urine 236 

38.  Ehrlich's  Copper  Plate 257 

39.  Favus  Fungi 309 

40.  Hair  in  Sycosis 313 

41.  Epidermal  Scales 314 

42.  Spirochetse 317 

43.  Migrating  Cells  and  Papillary  Bodies  . .  .325 


LIST  OF  COLORED  PLATES 

REFERRED  TO 

PLATE  FIG.  ON  PAGE 

I,  A.    Diphtheria  Bacilli 3 

I,  B.     Diphtheria  Bacilli 3 

II,  C.     Sputum  with  Typhoid  Bacilli 40 

II,  D.     Pneumonic  Sputum 48 

III,  E.     Pneumonic  Sputum 48 

III,  F.     Bronchial  Sputum 51 

IV,  G.     Influenza  Bacilli 51 

V,  H.     Cholera  Dejection 120 

V,  I.     Uric  Acid 211 

VI,  J.     Ammonium  Urate 213 

VII,  K.  Nephritis  in  Pregnancy— Hematoidin  Crystals.  222 

VIII,  L.     Casts  from  Icteric  Urine 230 

IX,  M.     Leucocytes 227 

IX,  N.     Red  Blood-Corpuscles 229 

X,  O.     Hyaline  Casts 230 

X,  P.     Spermatozoa 235 

XI,  Q.     Echinococcus  Booklets 235 

XI,  R.     Bacterium  Coli 239 

XII,  S.     Tubercle  Bacilli 240 

XII,  T.     Gonococci 247 

XIII,  U.     Macrocytes,  Microcytes,  etc 262 

XIII,  V.     Tertian  Parasite 265 

XIV,  W.     Tertian  Parasite ....  265 

XIV,  X.     Chronic  Tropical  Malaria 267 

XV,  Y.     Spirilla  of  Relapsing  Fever 268 

XV,  Z.     Anthrax  Bacilli  in  Rabbit's  Blood 301 

XVI,  a.  Tetanus  Bacilli..                                                     .302 


CHAPTER  I 

BACTERIOLOGICAL  EXAMINATION  OF  THE 

SECRETIONS  AND  DEPOSITS  IN  THE 

MOUTH  AND  PHARYNX 

In  the  bacteriological  examination  of  pathological 
products  found  in  the  mouth  and  pharynx,  the  place  of 
first  importance  belongs  to  the  diphtheria  bacilli.  Of 
secondary  importance  only  are  the  staphylo-,  strepto-, 
pneumo-cocci,  influenza  bacilli,  and  the  diplobacillus  of 
Friedlaender  j  which  appear  as  independent  exciters  of 
inflammation  in  angina,  as  well  as  producers  of  mixed 
infection  in  diphtheria.  Finally,  the  "soor  fungus," 
Of  (Hum  albicans,  should  be  mentioned.  In  coatings  on 
the  tonsils,  which  are  excited  by  the  Oidium  albicans, 
diphtheria  bacilli  are  also  not  infrequently  found. 

Collection  of  Material  to  be  Examined 

For  the  collection  of  material  from  the  mouth  and 
pharynx  it  is  best  to  use  the  small  apparatus  which  can 
be  obtained  from  any  supply  station.  This  consists  of  a 
strong  test-tube  containing  a  piece  of  wire,  one  end  of 
which  is  placed  in  the  plug  which  closes  the  tube,  while 
the  other  carries  a  swab  of  common  cotton.  The  test-tube 
and  contents  are  sterilized  by  dry  heat  for  half  an  hour  at 
a  temperature  of  160°  C.,1  and  then  placed  in  a  suitable 
box,  containing  directions  for  use  and  a  blank  to  be  filled 
out  by  the  physician,  stating  duration  of  illness,  locality 
from  which  the  material  to  be  examined  is  taken,  etc. 

1  All  degrees  of  temperature  quoted  in  this  book  are  Celsius. 

1 


S     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

To  obtain  the  material  for  examination,  the  swab  is 
brushed  firmly  across  the  suspected  coating  and  returned 
immediately  to  the  test-tube  (care  being  taken  that  it 
touches  nothing  but  the  coating  in  question).  No  anti- 
septic (gargle  or  application)  should  be  used  for  some 
time  before  the  collection  of  the  material,  as  the  growth 
of  the  diphtheria  bacillus  is  inhibited  by  even  mild  anti- 
septics. 

Morphological  and  Staining  Characteristics  of 
Diphtheria  Bacilli 

Diphtheria  bacilli  are  non-motile  rods  which  show 
differences  in  their  morphological  characteristics  depend- 
ing upon  the  nature  of  the  culture  media,  the  age  of  the 
culture,  and  the  temperature  at  which  they  are  cultivated. 
They  vary  considerably  in  length,  so  that  short,  medium, 
and  long  forms  may  be  differentiated.  A  six  to  ten  hours' 
growth  on  pure  serum,  or  Loeffler's.  blood  serum,  consists 
principally  of  long,  partly  straight,  partly  slightly  curved 
bacilli,  club-shaped  or  pointed  at  both  ends.  In  older 
cultures  the  spindle,  dumb-bell,  and  lancet  forms  are  seen. 
Small,  highly  refractive  points  can  be  seen  in  the  proto- 
plasm of  the  long  forms.  The  formation  in  colonies  of 
the  diphtheria  bacilli,  of  loosely  arranged  groups,  in  which 
the  individual  bacilli  lie  crosswise  over  each  other,  is 
characteristic.  Especially  in  A7«/scA-preparata  from 
young  serum  cultures  and  in  membranes  in  which  the 
diphtheria  bacilli  are  present  in  great  numbers,  a  picture 
is  seen,  which  may  be  imitated  by  holding  the  outspread 
fingers  of  one  hand,  in  various  combinations,  over  or  be- 
side those  of  the  other  (Neisser). 

Diphtheria  bacilli  stain  easily  with  dilute  aniline 
dyes.  Dilute  ZieliPs  solution  (1:9),  and  Loefflcr's  methy- 
lenp.  blue,  are  especially  adaptable.  The  former  stains  in 


MOUTH   AND   PHARYNX  3 

one  minute,  the  latter  in  two  minutes,  without  heating. 
Diphtheria  bacilli  stain  according  to  Gram.  Bacilli  from 
young  cultures  stain  evenly,  while  in  smears  from  older 
cultures  they  usually  show  one  or  more  unstained  spots. 
Bacilli  stained  with  Loeffler's  methylene  blue,  as  a  rule, 
show  either  at  one  or  both  ends,  granules  more  deeply 
stained  than  the  rest  of  the  protoplasm.  These  polar 
granules  (Babes- Ernsts  bodies)  are  especially  distinct  in 
specimens  stained  according  to  the  Roux  or  Neisser 
methods  (Plate  I,  Fig.  A).  Roux' s  solution  (cf.  p.  333) 
is  made  by  mixing  1  part  dahlia-violet  solution  with  2 
parts  methyl-green  solution.  The  mixture  keeps  well  and 
produces  no  precipitate.  It  stains  in  two  minutes  without 
heating.  Neisser's  is  a  double  staining  method  (formula 
cf.  p.  333). 

1.  Old  Method. — Stain  with  acetic  acid  methylene  blue 
twenty   seconds,   wash  with  distilled  water,  counterstain 
with  Bismarck  brown  ten  seconds,  wash,  etc. 

2.  New  Method. — Stain  with  Solution  I  (cf.  formulae 
of  stains)  for  about  thirty  seconds,  wash  with  distilled 
water,  counterstain  with  Solution  II,  also  for  about  thirty 
seconds.     The  bacilli  then  appear  brown,  the  oval  polar 
granules  dark  blue  (Plate  I,  Fig.  B).     As  a  rule,  each 
bacillus  shows  two  granules,   one  at   each  end.     Some, 
however,  have  but  one,  at  one  end,  while  still  others  have 
a  third  in  the  middle.     It  is  common  to  find  two  bacilli 
at  an  obtuse  angle  to  each  other,  having  together  three  or 
four  granules.     This  method  of  staining  is  only  sure  of 
success  when  the  smears  are  made  from  serum  cultures, 
which  are  at  least  nine  and  not  more  than  twenty  to 
twenty-four  hours  old,  and  have  been  grown  at  a  tempera- 
ture of  34°  to  37°  C.     It  is   further  important  that  the 
smears  be  very  thin.     It  is  necessary  to  test  newly  made 
solutions  for  Neisser's  method,  as  the  staining  power  of 


4     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

different  solutions  varies.  Neisser's  method  is  of  value 
in  differentiating  between  diphtheria  bacilli  and  other 
bacilli  resembling  them. 

Cultural  Characteristics 

For  the  cultivation  of  diphtheria  bacilli  a  temperature 
of  at  least  20°  C.  is  necessary.  They  grow  best  between 
33°  and  37°  C.  They  flourish  on  all  the  usual  culture 
media  which  have  a  slight  or  distinct  alkaline  reaction. 
For  diagnostic  purposes  bouillon,  glycerine  agar  (5  to  7 
per  cent.),  and  especially  blood  serum,  are  used. 

After  one  to  two  days'  growth,  bouillon  is  either  evenly 
clouded,  with  flakes  in  it,  or  a  fine  granular  precipitate 
has  developed,  which  collects  on  the  sides  and  bottom  of 
the  glass.  Not  infrequently  a  thin,  granular,  and  easily 
destroyed  film  forms  on  the  surface  of  the  bouillon.  The 
bouillon,  originally  faintly  alkaline,  becomes  acid  after 
forty-eight  hours'  growth  of  diphtheria  bacilli  in  it.  The 
growth  on  agar  is  scanty,  but  that  on  glycerine  agar  is 
richer.  The  surface  colonies  appear  transparent  and  gray- 
ish-white; examined  with  low  power  they  show  a  charac- 
teristic granular  surface  and  an  irregular  delicate  border. 
The  most  luxuriant  and  quickest  growth  of  diphtheria 
bacilli  is  that  on  blood  serum.  For  their  cultivation  from 
membranes  it  is  especially  suitable,  as  it  has  a  selective 
action  against  the  other  bacteria  of  the  mouth.  On  blood 
serum  the  diphtheria  bacilli  grow  first,  while  other  organ- 
isms, which  may  at  the  same  time  be  present,  develop 
later. 

Either  pure  blood  serum,  which  may  come  from  cattle, 
sheep,  or  horses  is  used,  or  the  so-called  Loeffle^s  serum, 
which  is  a  mixture  of  3  parts  calf  or  sheep  serum  with  1 
part  of  1  per  cent,  grape-sugar  bouillon  (cf.  p.  354). 

On  solidified  serum  the  diphtheria  bacilli  have  often, 


MOUTH   AND   PHARYNX  5 

after  but  six  hours'  growth,  developed  very  small  trans- 
parent colonies.  After  twenty- four  hours'  growth  the  col- 
onies are  about  the  size  of  a  pin's  head,  round,  prominent, 
and  yellowish-white.  If  the  colonies  lying  close  together 
coalesce,  a  yellowish-white  coating  is  formed,  which  still 
presents  a  distinctly  granular  appearance. 

Animal  Inoculation 

Inoculation  of  guinea-pigs  is  used  for  diagnostic  pur- 
poses as  a  means  of  differentiating  the  diphtheria  bacilli 
from  bacteria  resembling  them.  Guinea-pigs  weighing 
200  to  800  grammes  are  inoculated  subcutaneously  with 
2  cc  of  a  forty-eight  hours'  bouillon  culture.  The  animals 
become  sick  quickly.  After  twelve  to  twenty-four  hours 
a  distinct  infiltration  can  be  felt  at  the  point  of  inocula- 
tion; after  two  to  three  days  the  animal  dies.  The  post 
mortem  shows  at  the  site  of  inoculation  a  jellylike,  cedem- 
atous,  blood-stained  swelling  of  the  subcutaneous  tissue. 
The  peritoneal  and  pleural  cavities  contain  serous  and  fre- 
quently hemorrhagic  'exudates.  The  condition  of  the  ad- 
renal bodies  is  characteristic.  They  are  enlarged  and 
hyperaemic,  and  their  tissue  is  permeated  by  small  punc- 
tiform  hemorrhages.  In  the  cedema  fluid  at  the  site  of 
inoculation  diphtheria  bacilli  can  usually  be  detected  by 
cultural  methods. 

If  the  bacilli  were  less  virulent,  the  animal  dies  later, 
and  the  post-mortem  appearances  are  not  so  typical.  If 
still  less  virulent,  merely  a  local  inflammation  at  the  site 
of  inoculation  is  produced,  which  causes  necrosis  of  the 
skin  and  eventually  heals. 

Differential  Diagnosis 

In  the  differential  diagnosis  pseudo-diphtheria  bacilli 
(Hoffmann}  and  xerosis  bacilli  come  into  consideration. 


6     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

The  former  are  among  the  normal  inhabitants  of  the 
mouth,  the  latter  are  found  on  the  conjunctiva  and  in  the 
nose.  The  morphological  differences  between  pseudo-  and 
true  diphtheria  bacilli  are  most  evident  in  Klatsch-prQ- 
parata  from  young  (six  to  ten  hours')  blood-serum  cul- 
tures. Here  the  pseudo-diphtheria  bacilli  appear  usually 
as  short,  plump,  often  wedge-shaped  rods ;  the  characteris- 
tic long  forms,  which  diphtheria  cultures  of  the  same  age 
show,  are  missing.  The  typical  grouping  of  the  bacilli  is 
also  lacking.  Pseudo-diphtheria  bacilli  lie  usually  with 
the  long  sides  parallel,  side  by  side,  arranged  like  pali- 
sades. In  smears  from  older  (sixteen  to  twenty-four 
hours')  cultures  the  differentiation  may  be  difficult. 

Pseudo-diphtheria  bacilli  differ  from  true  diphtheria 
bacilli,  in  their  staining  properties,  by  their  failure  to 
stain  according  to  Neisser^s  method.  Although  some- 
times pseudo-diphtheria  bacilli,  if  stained  by  this  method 
show  pole  kernels,  they  are  always  found  but  scarcely, 
never  as  regularly  as  in  diphtheria  bacilli.  It  is  best  to 
use  cultures  of  from  twelve  to  twenty- four  hours  for  the 
differential  diagnostical  staining,  because  the  absence  of 
the  pole  kernels  in  cultures  of  from  six  to  twelve  hours 
means  just  as  little  as  the  presence  in  older  colonies. 

In  their  cultural  characteristics  they  differ  by  their 
luxuriant  growth  on  agar  and,  at  first,  slower  development 
on  serum.  The  colonies  are  grayish-white,  moistly  glis- 
tening, and  somewhat  waxy  in  appearance.  It  is  remark- 
able what  soft  and  melting  consistency  they  show,  when 
touched  with  the  platinum  needle  in  contradistinction  to 
the  more  rigid  diphtheria  bacillus  colonies. 

The  faculty  of  diphtheria  bacilli  of  forming  acid  in 
culture  media  containing  sugar  may  also  be  of  differential 
diagnostical  value. 

Diphtheria  bacilli  decompose    dextrose  and  levulose 


MOUTH   AND   PHARYNX  7 

under  acid  formation,  pseudo-diphtheria  bacilli,  as  proven 
in  numerous  types  of  various  origin  are  almost  always  in- 
active  in  both  kinds  of  sugar,  and,  but  in  rare  cases,  they 
are  active  in  one,  never  in  both. 

The  following  culture  media  are  used  for  testing  acid 
formation:  Three  parts  of  ox  serum,  which  is  rendered 
sterile  or  made  germ  free  by  discontinuing  sterilization  at 
55°,  are  added  to  one  part  of  sterile  bouillon,  which  is 
free  of  sugar.  To  each  90  parts  of  this  mixture  are  added 
10  parts  of  a  litmus  solution,  which  contains  10  per  cent, 
dextrose,  resp.  levulose,  in  order  to  obtain  2  media,  of 
which  one  contains  1  per  cent,  dextrose,  the  other  1  per 
cent,  levulose.  The  litmus  sugar  solution  before  being 
added,  has  to  be  sterilized  on  two  consecutive  days,  for 
five  minutes  each  day.  The  culture  media  are  then  filled 
in  test-tubes  and  are  kept  on  three  to  five  consecutive  days, 
for  two  hours  each  day,  in  the  incubator  at  55°. 

Procedure. — A  test-tube  of  levulose  and  a  test-tube  of 
dextrose  are  each  inoculated  with  a  loop  of  the  pure  cul- 
ture to  be  examined.  After  twenty-four  hours  in  the  in- 
cubator at  87°  the  litmus  solution  appears  red  in  the  test- 
tubes,  which  have  been  inoculated  with  diphtheria  bacilli 
and  the  serum  albumin  is  precipitated. 

The  test-tubes  inoculated  with  pseudo-diphtheria  bacilli 
as  a  rule  appear  unchanged,  only  sometimes  a  little  acid 
formation  can  be  traced. 

The  same  result  is  also  obtained  from  nutrose  litmus 
bouillon : 

Liebig's  meat  extract, 

Pepton  .         .         .  aa       2.0 

Aq.  dest         ....   150.0 

Boil  in  a  steam-pot  until  the  pepton  is  dissolved,  then 
neutralize  with  a  10  per  cent,  soda  solution,  again  boil 


8     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

up  and  filter.  Then  are  added  a  solution  of  2.0  nutrose 
in  50  cc  water,  2.0  dextrose  or  levulose  and  20  cc  litmus 
solution  (Kalilbaum) .  After  precipitation  in  sterile 
test-tubes  the  culture  medium  is  again  sterilized  by  steam 
on  three  consecutive  days,  fifteen  minutes  each  time. 

Xerosis  bacilli  resemble  in  appearance  the  long  forms 
of  the  diphtheria  bacilli,  but  produce  a  slower  growth  on 
serum  and  glycerine  agar.  After  six  hours'  growth  on 
blood  serum  they  are  so  slightly  developed,  and  cling  so 
tightly  to  the  culture  media,  that  in  A7#/sc7i-preparata  no 
typical  groups  appear.  Even  after  twenty  hours  the  devel- 
opment of  colonies  is  not  marked,  and  still  allows  the 
making  of  ./Tfefcc/j-preparata,  which,  with  true  diphtheria 
bacilli,  is  no  longer  possible  because  of  their  luxuriant 
growth. 

It  is  not  always  possible  to  distinguish  xerosis  bacilli 
from  true  diphtheria  bacilli  by  Neisser's  method,  as 
xerosis  bacilli  also  frequently  show  polar  staining.  In 
relation  to  acid  formation  in  culture  media  of  dextrose 
and  levulose  they  react  like  pseudo-diphtheria  bacilli. 
Animal  inoculation  is  the  surest  means  of  differentiation, 
as  xerosis  bacilli  show  themselves  non-virulent. 

Order  of  Examination 

Four  unused  cover  glasses,  sterilized  in  the  flame,  are 
smeared  with  the  material  to  be  examined.  The  smears 
are  stained  with  ten  times  diluted  ZiehVs  solution,  with 
Loeffler's  methylene  blue,  according  to  Roux  and  accord- 
ing to  Gram.  Cultures  are  also  planted  upon  blood 
serum  and  glycerine  agar. 

Examination  of  the  Smears. — In  some  cases,  even  in 
the  smears,  numerous  bacilli  are  seen  which,  both  in  form 
and  position,  resemble  diphtheria  bacilli,  so  that  from 
this  fact  alone  a  probable  diagnosis  of  diphtheria  may  be 


MOUTH  AND  PHARYNX  9 

made :  the  result  of  the  cultures  must,  however,  be  awaited 
before  giving  a  definite  report.  In  a  very  large  number 
of  cases,  however,  only  a  few  isolated  suspicious-looking 
bacilli  are  found,  or  often  these  are  missing.  Diplo-, 
strepto-,  staphylococci,  bacilli  which  do  not  stain  by 
Gram,  spirilli,  etc.,  are  present.  Nevertheless,  the  case 
may  be  one  of  diphtheria,  as  the  result  of  the  cultures 
will  later  show. 

Examination  of  the  Plates. — .fftosc/a-preparata  are 
made  from  the  serum  plates  after  they  have  been  six 
hours  in  the  incubator,  and  are  stained  with  fuchsin  or 
Loeffler's  methylene  blue.  If  the  above  described  bacilli 
arranged  in  typical  groups  are  found,  the  diagnosis  of 
diphtheria  is  very  probable.  After  ten  to  eighteen  hours' 
growth  the  plates  are  again  tested.  If  the  material  under 
examination  contained  diphtheria  bacilli  capable  of  devel- 
opment, they  will  by  this  time  have  developed  the  charac- 
teristic colonies  in  nearly  pure  culture.  Smears  are  now 
made  from  the  serum  plates  and  stained  with  Loefflcr's 
methylene  blue,  and  according  to  Roux  and  Neisser.  If 
these  contain  almost  exclusively  bacilli  which  show  the 
characteristic  polar  staining,  the  diagnosis  of  diphtheria 
can  be  made,  provided  that  the  material  examined  came 
from  a  sick  person  and  ivas  taken  from  the  pharynx. 

If  after  twelve  to  twenty-four  hours  cocci  alone  have 
grown,  it  is  very  improbable  that  the  case  is  one  of  diph- 
theria; however,  the  plates  must  be  examined  again  on 
the  following  day,  as  in  rare  cases  diphtheria  bacilli  de- 
velop late — namely,  when  gargles,  etc.,  have  been  used 
shortly  before  obtaining  the  material  for  examination. 

Colonies  of  diphtheria  bacilli  can  also  be  seen  on 
glycerine  agar  plates  after  twelve  hours'  growth  at  37°  C. ; 
but  the  other  micro-organisms  which  were  present  in  the 
material  have  also  developed  by  this  time.  These  bac- 


10    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

teria  are  identified  according  to  the  methods  described 
under  Examination  of  Sputum. 

Oidium  Albicans—" SOOT  Fungus"  (Fig.  1) 

The  detection  of  the  "soor  fungus"  succeeds  best  in 
unstained  specimens,  made  by  teasing  a  particle  taken 


FIG.  1.— Oidium  Albicans. 


from  the  suspected  coating  in  a  drop  of  water  or  glycerine. 
In  such  specimens  are  seen  double-contoured  hyaline 
hyphse  (mycelia),  having  transverse  septa  and  indenta- 
tions, and  often  having  lateral  branches  which  interlace 
with  one. another.  Among  the  hyphse  (mycelia)  appear 
the  conidia,  sometimes  spherical,  sometimes  cylindrical. 


MOUTH   AND   PHARYNX  11 

The  specimens  may  be  stained  according  to  Gram  or 
Kuelme-Weigert  (cf.  p.  338). 

The  cultivation  of  the  fungus  is  unnecessary  for  diag- 
nosis. It  can,  however,  be  easily  grown  on  all  the  usual 
culture  media;  on  agar  it  produces  porcelainlike,  glisten- 
ing white  colonies,  composed  of  yeastlike  oval  cells,  and 
containing  only  isolated,  short  hyphae  (mycelia).  These 
develop  more  richly  in  gelatine  stab  cultures,  in  the  lateral 
branches  extending  from  the  stab  canal.  Gelatine  is  not 
liquefied. 

In  the  coatings  of  the  tonsils  which  are  produced  by 
the  oidium  albicans,  are  found  occasionally  at  the  same 
time  diphtheria  bacilli,  when  a  culture  is  made. 

Angina  Vincent!  (s.  Plautii) 

The  diagnosis  of  the  Plant-  Vincent  angina  is  made 
from  the  stained  smear.  But  owing  to  the  fact  that  this 
disease  is  sometimes  combined  with  diphtheria,  the  ma- 
terial must  always  be  inoculated  in  blood  serum. 

The  specimens  are  stained  with  a  diluted  ZiehTs  solu- 
tion (cf.  solutions  for  staining)  or  according  to  Giemsa. 

The  stained  specimens  show  that  they  are  composed 
of  coating,  which  is  easily  removable,  a  paste  of  a  grayish- 
brown  or  slightly  greenish  color  and  ill-smelling,  necrotic 
tissue,  which  contains  a  very  large  quantity  of  fusiform 
bacilli  and  mostly  numerous  spirochetse.  In  the  diph- 
theroid  or  pseudo-membranous  form  of  the  angina  Vin- 
cent i  as  a  rule  only  fusiform  bacilli  are  found,  in  the 
ulcerous  type  are  also  found  spirochetae.  In  fresh  cases 
the  bacilli  fusiformes  and  the  spirochetse  appear  almost  in 
pure  cultures,  whereby  only  a  few  of  the  ordinary  mouth 
bacteria  are  found.  Only  at  about  the  termination  of  the 
disease  the  accompanying  bacteria  are  brought  more  into 
the  foreground. 


12    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

The  bacilli  fusiformes  are  mostly  long,  slender  rods, 
pointed  at  their  ends,  have  a  slight  swelling  in  the  centre, 
and  therefore  appear  fusiform ;  they  are  straight  or  slightly 
curved  (comma-shaped).  In  the  stained  specimen  we  see 
in  the  centre  an  oval,  unstained  vacuole.  Besides  these 
typical,  long,  slender  forms  are  also  found  shorter  rods 
and  thin,  long,  threadlike  bodies,  pointed  at  their  ends, 
frequently  S-shaped.  In  the  smears  the  bacilli  fusiformes 
are  found  mostly  singly  spread  over  the  entire  visual  field, 
often  in  twos,  which  form  more  or  less  obtuse  angles; 
more  seldom  in  heaps,  in  which  case  their  formation 
resembles  the  typical  form  of  diphtheria  bacilli. 

According  to  Gram's  method  they  are  negative. 

Regarding  their  motility  the  views  are  divided.  Some 
authors  believe  them  to  be  immovable,  others  say  that 
they  move  but  slowly,  Gr  emptier  could  see  active  motility, 
although  quickly  diminishing. 

We  can  culture  the  bacilli  fusiformes  only  anaerobic 
in  serum  or  ascites  agar  at  87°.  After  twenty-four  to 
forty-eight  hours  we  can  see  fine  yellowish-white  colonies, 
which  have  a  somewhat  darker  centre  with  radiations  in 
all  directions.  The  cultures  have  a  fetid  odor. 

The  spirochetse  which  in  most  of  the  cases  accompany 
the  bacilli  fusiformes,  look  the  same  as  the  spirochetse 
which  we  find  ordinarily  in  the  oral  cavity,  especially  in 
the  dental  deposit  (according  to  Muelilens,  spirocheta  buc- 
calis  and  the  middle  form  of  the  mouth  spirochetse.  They 
are  corkscrew-shaped,  very  motile  in  shape  and  size, 
however,  very  much  different  from  each  other.  We  find 
together  thin  and  thick  spirochetse,  some  which  have  three 
to  five  windings,  and  longer  ones  with  ten  and  more 
windings.  Most  of  them  are  flat  and  irregular;  they 
straighten  out  only  while  in  motion.  The  windings  of 
others  again  show  resemblance  to  the  spirocheta  pallida. 


MOUTH   AND   PHARYNX  13 

(Examination  of  the  fresh  specimen  with  the  diaphragm 
shut. ) 

The  spirochetae  do  not  stain  so  readily  as  the  bacilli 
fusiformes;  they  are  also  negative  according  to  Gram's 
method.  They  are  found  in  the  smears,  isolated  or  in 
more  or  less  thick  heaps  and  entwined  in  each  other,  like 
in  nests. 

The  culture  of  the  mouth  spirochetse  can  be  obtained 
in  pure  cultures  under  anaerobic  conditions  in  serum  agar 
(MueJilens) .  Not  before  eight  to  ten  days  appear  very 
fine  colonies  at  the  bottom,  which  render  the  culture  media 
cloudy.  The  cultures  have  a  fetid  odor. 

Stomatitis  Ulcerosa. — The  specimens  which  are  stained 
from  the  coverings  of  the  ulcers  are  identical  with  those 
in  angina  Vincent i. 

Memngococci. — The  meningococci  are  found  in  the  mu- 
cus of  the  naso-pharyngeal  cavity  in  patients  suffering 
from  epidemic  cerebro-spinal  meningitis,  especially  in  the 
beginning  of  the  disease  and  in  persons  who  have  come  in 
contact  with  such  patients. 

The  specimen  is  to  be  taken  from  the  upper  part  of 
the  naso-pharyngeal  cavity  around  the  pharynx  tonsil 
by  way  of  a  right-angularly-upward-bent  probe,  which 
is  put  in  the  mouth  and  then  upward  behind  the  soft 
palate. 

Morphological  and  Tinctorial  Properties. — The  menin- 
gococci are  diplococci  which  resemble  gonococci  in  shape 
and  adjustment.  The  frequent  presence  of  tetrades  is 
characteristic  for  specimens  and  cultures.  The  individual 
cocci  often  differ  in  size.  Sometimes  very  large  specimens 
are  found,  besides  normal  and  well-stained  cocci,  and  often 
cocci  three  times  the  size  smaller  and  poorly  stained. 
This  shows  especially  in  older  cultures  after  about  forty- 
eight  hours.  In  the  secretions  sometimes  the  meningo- 


14    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

cocci  are  found  grouped  in  small  heaps  within  the  pus 
cells. 

According  to  Gram's  method  they  are  negative,  the 
same  as  the  gonococci  and  the  micrococcus  catarrhalis. 

Cultural  Properties. — The  meningococci  develop  best 
in  ascites  agar  (one  part  ascites  fluid  plus  three  parts 
agar)  at  37°.  They  also  grow  very  well  in  Loeffler's  blood 
serum  (cf.  culture  media). 

The  meningococci  do  not  grow  in  ordinary  agar  in  the 
first  generation,  but  they  get  accustomed  to  this  medium, 
especially  when  frequently  transplanted  in  later  genera- 
tions. After  twenty-four  hours'  growth  in  ascites  agar 
colonies  are  formed  of  2  to  4  mm,  which  have  a  grayish- 
white  appearance,  but  show  a  lustre  of  mother-of-pearl  by 
transmitted  light.  Under  the  microscope  they  are  grayish- 
yellow,  smooth-edged,  homogeneous  or  indistinctly  granu- 
lated. Two  distinct  zones  are  in  older  colonies,  a  slightly 
elevated,  central  zone,  and  a  flat,  peripheral.  On  the 
sloping  surface  of  ascites  agar  a  homogeneous,  grayish 
colony  is  formed,  ascites  bouillon  becomes  cloudy,  and 
sometimes  a  top  skin  is  formed.  The  meningococci  do 
not  coagulate  milk  nor  do  they  grow  much  in  it.  They 
transform  dextrose  and  maltose  into  acids,  but  they  can- 
not attack  levulose,  mannit,  milk-sugar,  cane-sugar,  dul- 
cit,  galactose  and  inulin.  v.  LingelsJieim  prepared  an 
ascites  litmus  sugar  agar  (cf.  culture  media)  to  ascertain 
this  reaction  of  the  meningococci.  The  medium  contain- 
ing maltose  or  dextrose  is  stained  red  through  the  growth 
of  the  meningococci,  if  the  other  kinds  of  sugar  (levulose, 
etc. )  are  added,  the  medium  remains  blue.  For  diagnosis 
the  test  with  maltose,  dextrose,  and  levulose  is  sufficient. 
The  cultures  are  not  very  resistible.  They  must  be  kept 
at  87°,  and  they  must  be  transplanted  in  a  new  raedium 
— at  first  daily,  later  every  six  to  seven  days. 


MOUTH  AND   PHARYNX  15 

The  animal  test  is  of  no  diagnostic  value. 

The  agglutination  test.  By  immunizing  rabbits,  and 
especially  horses,  with  meningococci,  a  serum  is  obtained 
which  agglutinates  most  of  the  species,  which  have  the 
characteristic  properties  of  the  meningococci,  but  some  of 
them  are  not  so  easily  agglutinable,  and,  therefore,  they 
cannot  be  differentiated  by  the  agglutination  test.  It  is 
best  to  take  cultures  of  forty-eight  hours'  growth.  The 
inoculated  test-tubes  must  be  kept  standing  for  twenty- 
four  hours  at  55°,  covered  with  a  rubber  cap,  before  ascer- 
taining the  result. 

Differential  Diagnosis. — The  differential  diagnosis  is 
to  be  made  against  the  gonococcus  and  a  number  of  Gram 
negative  diplococci,  which  are  found  in  inflammatory 
affections  of  the  bronchi  and  also  in  the  normal  mucus  of 
the  pharynx  (micrococcus  catarrhalis,  diplococcus  flavus 
species,  micrococcus  pharyngis  cinereus). 

It  is  impossible  to  make  a  distinction  between  these 
diplococci  in  the  stained  specimen,  only  by  their  cultural 
properties  and  by  the  agglutination  test.  The  'latter  does 
not  hold  good  in  gonococci,  because  the  gonoco&ci  are  ag- 
glutinated from  a  very  much  diluted  meningococcus  serum. 
The  other  diplococci  are  influenced  by  it  in  no  higher 
dilution  than  from  normal  rabbit  or  horse  serum.  Some- 
times they  are  agglutinated  spontaneously  in  salt  solutions. 
A  sure  differential  diagnosis  between  meningococci  and 
gonococci  can  be  made  from  cultures  only. 

The  colonies  of  the  gonococci  are  smaller  than  those  of 
the  meningococci,  they  do  not  coalesce;  they  are  clammy 
and  slimy,  and  generally  distinctly  granulated  at  a  low 
magnification.  Gonococci  only  grow  in  media,  which 
contain  human  albumin  in  a  non-coagulated  state;  menin- 
gococci develop  also  in  Loeffler's  blood  serum. 

The   micrococcus   catarrhalis   also    differs    from   the 


16    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

meningococci  by  its  cultures ;  besides  that  it  grows  already 
in  the  first  generation  in  ordinary  agar  and  is  unable  to 
attack  any  of  the  above-mentioned  kinds  *of  sugar. 

The  cultures  of  the  species  diplococcus  flavus  (according 
to  v.  Lingelslieim  there  are  three  species)  are  characterized 
by  the  formation  of  a  yellow  pigment. 

The  micrococcus  cinereus  differs  from  the  meningo- 
coccus  microscopically  by  its  coarse,  irregular,  occasionally 
oblong  texture  (v.  LingelsJieirti).  Its  colonies  look  like 
those  of  the  micrococcus  catarrhalis,  and  it  is  likewise 
unable  to  attack  any  of  the  above-mentioned  kinds  of 
sugar. 

The  examination  for  meningococci  must  be  made  as 
soon  as  possible  after  the  mucus  has  been  removed  from 
the  pharynx.  The  diagnosis  of  the  presence  of  meningo- 
cocci in  the  pharyngeal  secretion  can  only  be  established 
by  making  cultures  because  of  the  presence  of  the  above 
named  diplococci,  which  look  alike  microscopically.  The 
probe  to  which  the  mucus  adheres  is  smeared  over  three 
ascites  plates.  After  twenty-four  hours'  growth  at  37° 
the  suspicious-looking  colonies  are  stabbed  and  transported 
to  sloping  ascites  agar.  The  cultivated  pure  cultures  are 
identified  by  the  agglutination  test  and  by  their  cultural 
properties. 


CHAPTER   II 

BACTERIOLOGICAL  EXAMINATION   OF   NASAL 
SECRETIONS 

The  nasal  secretion  is  removed  for  examination  by 
means  of  a  swab  or  platinum  wire,  with  the  aid  of  a  head 
mirror. 

The  material  is  examined  in  stained  smears,  by  cultural 
methods,  and  by  animal  inoculation.  The  bacilli  which 
come  especially  into  consideration  are  diphtheria  bacilli, 
tubercle  bacilli,  lepra  bacilli,  influenza  bacilli,  and  the 
micro-organisms  belonging  to  the  group  of  the  diploba- 
cillus  of  Friedlaender — the  so-called  oza?na  and  rhinos- 
cleroma  bacilli. 

Diphtheria  Bacilli. — The  recognition  of  diphtheria 
bacilli  is  accomplished  in  the  same  manner  as  in  the  ex- 
amination of  pharyngeal  coatings.  However,  animal  in- 
oculation is  necessary  for  the  verification  of  the  cultivated 
bacteria  in  cases  in  which  the  diphtheria  has  not  spread 
from  the  pharynx  to  the  nasal  cavity,  because  of  the  es- 
pecially frequent  presence  in  the  nose  of  bacteria  resem- 
bling diphtheria  bacilli. 

Tubercle  Bacilli. — Tubercle  bacilli  are  detected  by  means 
of  stained  smears.  For  the  differential  diagnosis  between 
tubercle  bacilli  and  other  acid-fast  bacilli  which  may  be 
normally  present  in  the  nose,  animal  inoculation  must  be 
used.  Lepra  bacilli  can  often  be  distinguished  from 
tubercle  bacilli  by  their  characteristic  position  and  ar- 
rangement. 

17 


18     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Lepra  Bacilli. — Smears  are  made  from  the  secretion 
removed  from  the  nose  with  a  platinum  wire,  and  stained 
according  to  the  method  for  tubercle  bacilli  and  according 
to  Baumgarten  (cf.  p.  882  and  Examination  of  Sputum). 
Bacteria  belonging  to  the  group  of  the  diplobacillus  of 
Friedlaender  are  very  frequently  present  in  the  nose  of  a 
healthy  person.  The  micro-organisms  which  have  been 
detected  in  ozsena  and  rhinoscleroma  cannot,  with  cer- 
tainty, be  separated  from  the  diplobacillus  of  Friedlaender. 
In  their  morphological  and  cultural  characteristics,  as 
well  as  in  their  behavior  when  inoculated  into  animals, 
they  agree  almost  exactly  or  exactly  with  it;  such  varia- 
tions from  the  typical  as  they  do  occasionally  show,  are 
not  more  marked  than  those  which  may  be  seen  in  differ- 
ent cultures  of  the  diplobacillus  of  Friedlaender  itself. 
As  yet  the  attempt  to  differentiate  between  the  three  kinds 
of  bacteria  by  means  of  agglutination  tests  has  failed 
(Klemperer  and  Sclieyer).  Concerning  the  detection  of 
these  bacteria  and  influenza  bacilli,  cf.  Examination  of 
Sputum,  p.  22. 


CHAPTER  III 

BACTERIOLOGICAL  EXAMINATION  OF 
CONJUNCTIVAL  SECRETION 

Material  for  examination  may  be  obtained  by  means  of 
the  swabs  suggested  for  obtaining  material  from  the  throat. 
If  the  secretion  is  very  thin,  sterile  capillary  tubes  are 
used,  which,  after  the  material  is  obtained,  are  sealed  at 
both  ends  by  melting  in  the  flame.  If  the  material  is  to 
be  examined  at  the  bedside,  it  is  best  obtained  with  a 
sterile  platinum  wire. 

The  examination  is  usually  made  by  means  of  stained 
smears  and  cultural  methods.  Animal  inoculation  is  re- 
sorted to  only  when  diphtheria  or  tubercle  bacilli  are  sus- 
pected. 

Diphtheria  Bacilli. — Detection  is  accomplished  accord- 
ing to  the  method  described  on  p.  8.  In  the  differential 
diagnosis  xerosis  bacilli  must  be  borne  in  mind  (cf.  p.  8). 

Tubercle  Bacilli. — In  tuberculosis  of  the  conjunctiva 
tubercle  bacilli  may  occasionally  be  detected  even  in  the 
smears;  in  many  cases,  however,  animal  inoculation  is 
necessary.  As  material  for  inoculation,  the  secretion  from 
an  ulcer,  or  a  small  piece  excised  from  the  conjunctiva, 
may  be  used. 

Often  in  these  cases  the  injection  is  made  into  the  an- 
terior chamber  of  the  eye  of  a  rabbit.  The  animal  being 
fastened  on  the  operating  board  and  the  eye  anaesthetized 
with  10  per  cent,  cocain,  a  fold  of  conjunctiva  is  grasped 
with  the  thumb  forceps  and  the  eyeball  drawn  downward 

19 


20     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

and  incised  close  to  the  upper  edge  of  the  cornea.  To 
prevent  injury  to  the  iris,  the  lancet  must  be  introduced 
parallel  to  it,  and  withdrawn'  with  its  point  directed  toward 
the  cornea  (by  sinking  the  handle).  The  material  to  be 
inoculated  is  introduced  into  the  anterior  chamber  of  the 
eye  through  the  wound  on  the  upper  border  of  the  cornea, 
by  means  of  a  syringe  or  iris  forceps.  To  guard  against 
prolapse  of  the  iris,  a  solution  of  eserin  is  dropped  into 
the  eye  immediately.  During  the  next  few  days  a  solution 
of  atropin  and  cocain  should  be  dropped  into  the  eye  to 
allay  the  irritation  following  the  operation. 

Depending  upon  the  number  of  tubercle  bacilli  intro- 
duced, and  upon  whether  from  the  start  they  lay  free,  or . 
were  embedded  in  the  tissue,  there  develops,  after  one  or 
more  weeks,  a  tuberculosis  of  the  iris,  which  may  finally 
lead  to  a  caseous  phthisis  of  the  eyeball.  If  the  tubercu- 
losis spreads  farther,  it  attacks  first  the  neighboring  lymph- 
nodes  and  then  the  lungs,  and  eventually  produces  a  gen- 
eral tuberculosis,  which,  after  weeks  or  sometimes  months, 
causes  the  death  of  the  animal. 

Gonococci. — The  detection  of  gonococci  is  made  by 
means  of  smears  stained  with  dilute  methylene  blue  and 
by  Gram's  method.  For  their  identification  cultures  must 
be  made,  as  other  diplococci  are  found  in  the  conjunctival 
secretion  which  resemble  them  both  morphologically  and 
in  their  staining  characteristics  (cf.  Examination  of  Ure- 
thral  Secretion). 

Bacilli  of  Koch  and  Weeks. — These  are  found  as  the  ex- 
citers of  both  acute  and  chronic  conjunctivitis  in  the 
secretion  of  the  conjunctival  sac.  In  smears  made  from 
this  secretion  and  stained  with  dilute  borax  methylene 
blue,  especially  during  the  rise  and  height  of  the  conjunc- 
tivitis, though  also  in  chronic  cases,  numerous  fine  slim 
bacilli  of  varying  length,  resembling  influenza  bacilli,  are 


CONJUNCTIVAL   SECRETIONS  21 

found.  These  lie  either  within  the  pus  cells,  which  ap- 
pear stuffed  with  them,  or  outside  of  them.  They  are 
decolorized  by  Grain. 

The  bacilli  cannot,  as  a  rule,  be  cultivated  upon  plain 
agar.  They  flourish,  however,  on  human  blood  and  as- 
cites  agar,  and  develop,  after  twenty-four  to  forty-eight 
hours,  small  moist  colonies  resembling  dew-drops. 

Diplobacillus  of  Morax  and  Axenfeld. — Conjunctivitis 
excited  by  these  diplobacilli  produces  frequently  but  little 
secretion.  To  prepare  the  smears,  the  mucus  is  used, 
which  is,  though  in  small  amount,  usually  present  on  the 
caruncle.  In  smears  stained  with  dilute  methylene  blue 
the  bacilli,  which  in  appearance  resemble  the  diplobacilli 
of  Friedlaender,  are  seen,  some  within,  some  free,  and 
some  lying  upon  the  epithelial  cells.  They  are  usually 
arranged  in  twos,  and  present  themselves  as  plump  bacilli, 
resembling  a  rectangle  with  blunted  corners.  They  are 
decolorized  by  Gram. 

The  diplobacilli  grow  on  blood  serum  or  on  serum  agar. 
Blood  serum  is  liquefied,  and  after  twenty-four  hours  the 
surface  appears  uneven,  due  to  the  presence  of  small, 
moist,  somewhat  sunken  and  translucent  spots,  which 
gradually  become  deeper  and  deeper  (Axenfeld}.  In  pure 
cultures  involution  forms  develop — partly  grotesque,  partly 
very  large — even  after  but  two  days'  growth. 

Influenza  bacilli,  pneumo-,  strepto-,  staphylo-,  and 
meningococci  are  also  found  in  the  conjunctival  secretion 
as  exciters  of  conjunctival  catarrh.  Concerning  the  de- 
tection of  these  bacteria,  compare  Examination  of  Sputum 
and  Examination  of  Fluids  Obtained  by  Puncture. 


CHAPTER  IV 
EXAMINATION  OF  THE  SPUTUM 

Method  of  Obtaining  Material  for  Examination 

The  sputum  must  be  collected  in  a  clean  vessel.  It  is 
best  that  the  vessel  be  sterile,  and  that  the  sputum  be  ex- 
amined as  soon  as  expectorated.  When  this  is  not  possi- 
ble, it  is  well  to  collect  the  sputum  in  a  1  to  2  per  cent, 
carbolic  acid  solution,  which  has  been  proved  to  be  suffi- 
cient to  check  further  bacterial  development.  More  con- 
centrated carbolic  acid  solutions  are  to  be  avoided,  as  they 
render  the  sputum  unfit  for  examination.  Such  sputum 
cannot,  of  course,  be  used  for  cultural  tests. 

The  patient  should  be  instructed  to  bring  only  such 
sputum  for  examination  as  has  been  really  raised  by 
coughing,  and  not  by  hawking.  To  prevent  contamina- 
tion, the  mouth  should  be  rinsed  several  times  with  freshly 
boiled  water  before  expectoration.  When  the  expectora- 
tion is  slight,  it  is  best  to  examine  the  morning  sputum, 
or  when  it  is  a  question  of  determining  the  presence  of 
tubercle  bacilli,  to  collect  the  expectoration  of  the  entire 
day  in  a  vessel  which  can  be  tightly  closed,  and  which 
contains  1  to  2  per  cent,  carbolic  solution.  To  excite  ex- 
pectoration potassium  iodide  may  be  administered,  or 
moist  compresses  may  be  bound  over  both  shoulders  dur- 
ing the  night,  followed  in  the  morning  by  a  cold  rub 
down.  The  coughing  excited  by  the  shock  will  expel  the 
excretion  which  has  collected  under  the  influence  of  the 
moist  warmth. 

22 


SPUTUM  23 

General  Characteristics 

The  macroscopical  examination  of  the  sputum,  which 
should  always  precede  the  microscopical,  discloses  its  gen- 
eral characteristics.  For  this  purpose  the  sputum  is 
poured  into  a  flat  glass  dish,  the  so-called  Petri  dish,  and 
examined  over  a  dark  background.  Notice  should  be 
taken  of  the  quantity,  odor,  stratification,  color,  and  con- 
sistency of  the  sputum,  and  any  especially  prominent 
ingredients. 

Quantity  of  Sputum. — Though  this  varies  exceedingly  in 
the  majority  of  the  diseases  of  the  respiratory  tract,  yet 
for  certain  of  them  the  large  amount  of  sputum  produced 
is  in  itself  characteristic.  For  example,  a  noticeably  large 
quantity  is  expectorated  in  cases  of  empyema,  which  has 
ruptured  into  the  lungs,  and  in  bronchiectasis,  pulmonary- 
gangrene,  and  abscess. 

Odor. — Freshly  expectorated  sputum  has  usually  no 
characteristic  odor.  It  is  foul-smelling  only  when  it  has 
decomposed  as  the  result  of  long  standing.  Sputum  in 
diseases  in  which  its  decomposition  has  taken  place  with- 
in the  body  has  even  at  the  time  of  its  expectoration  a 
repugnant,  often  a  cadaverous  foul  odor  (pulmonary  gan- 
grene, putrid  bronchitis,  etc.). 

Stratification. — In  bronchiectasis,  putrid  bronchitis, 
and  pulmonary  gangrene,  the  sputum  separates  shortly 
after  its  expectoration  •  into  three  strata :  an  upper  foamy 
stratum,  greenish-yellow  in  color;  a  middle  translucent, 
serous  stratum,  and  a  lower  non-transparent  stratum,  puru- 
lent in  character.  Sputum  in  cases  of  lung  abscess  shows, 
usually  after  standing  some  time,  two  strata:  an  upper 
serous  stratum,  representing  the  pus  serum,  and  a  lower 
non-transparent  yellow  one,  containing  the  cellular  ele- 
ments. 


24    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Color. — The  color  depends  generally  upon  the  richness 
of  the  sputum  in  cellular  elements.  It  is  light  and  glassy 
in  sputum  containing  few  cells,  non-transparent  or  yellow 
in  that  containing  many.  The  color  caused  by  the  pres- 
ence of  blood  is  very  striking.  It  is  bright  red  and  foamy 
in  hemorrhages  from  eroded  bloodvessels ;  its  rusty  color 
in  pneumonia  is  pathognomonic ;  it  is  dark,  nearly  black 
in  cases  of  hemorrhagic  infarct  and  at  the  termination  of 
pthisical  hemorrhage.  The  thin  liquid  sputum  in  oedema 
of  the  lungs  is,  in  proportion  to  the  amount  of  blood  con- 
tained, yellow,  rose-colored,  or  dark  red ;  in  inflammatory 
oedema  complicating  croupous  pneumonia,  it  resembles 
prune  juice.  The  expectoration  in  hemoptysis  resulting 
from  neoplasms  may  resemble  currant  jelly.  When  spu- 
tum containing  blood  has  decomposed,  as  in  pulmonary 
gangrene,  it  has  a  brown  or  dirty  green  color.  Sputum 
may  be  stained  green  by  pigment  produced  by  bacteria 
(B.  pyocyaneus,  B.  fluorescens,  sarcince,  etc.). 

Composition  of  Sputum 

A  distinction  is  made  between  mucous,  muco-purulent, 
purulent,  and  bloody  sputa. 

I.  Mucoid  Sputum. — This  may  be  pure  mucoid  or  watery 
mucoid.     The  pure  mucoid  sputum  is  translucent,  gray- 
ish-white in  color,   tough  and  thready   in   consistency. 
The  watery   mucoid  sputum   is  more  liquid,   less  tough 
than  the  pure  mucoid,  and  frequently  so  rich  in  air-bub- 
bles that  the  entire  expectoration  is  covered  with  foam. 
The  mucoid  portions  lie  as  flakes  or  balls  in  the  deeper 
liquid  substance. 

II.  Muco-Purulent  Sputum.— This  may  be  muco-purulent 
and   nearly   homogeneous,  or   purulo-mucoid  and   non- 
homogeneous.     In  the  first  case,  the  sputum  forms  a  prac- 
tically homogeneous  non-transparent  mass  of  yellowish- 


SPUTUM  25 

white  appearance  and  of  still  comparatively  tenacious, 
gluey  consistency.  Only  by  examination  in  direct  light 
can  the  translucent  mucoid  portions  be  clearly  distin- 
guished from  the  purulent.  The  latter  permeate  in  streaks 
the  mucoid  mass.  The  fine  mixture  of  pus  and  mucus 
points  to  the  fact  that  both  were  produced  in  the  same 
portion  of  the  respiratory  tract. 

In  the  purulo-mucoid,  non-homogeneous  sputum  the 
purulent  outweigh  the  mucoid  ingredients.  The  purulent, 
greenish-yellow,  non-transparent  portions  are  not  mixed 
with  the  mucous,  but  build  either  round  nummular  discs 
(sputum  rotundum),  or,  after  longer  standing,  flow  to- 
gether, sink  to  the  bottom,  and  produce  stratified  sputum. 

III.  Purulent  Sputum. — This  is  greenish-yellow  in  color, 
homogeneous  in  appearance,  and  thickly  liquid  in  consist- 
ency.     Its  characteristic  division  into  strata   has  been 
mentioned. 

IV.  Bloody  Sputum. — 1.  Pure  bloody  sputum  (hemop- 
tysis is  either  liquid,  bright  red  and  foamy,  or  has  coag- 
ulated before  its  expectoration,   forming  thick  clumps. 
The  question  of  the  source  of   the  blood  is   occasionally 
difficult  to  answer ;  from  the  appearance  of  the  blood  alone, 
differential  diagnosis  between  hemoptysis  and  hemateme- 
sis  cannot  be   made.     Blood  coming  from  the  stomach 
has,  to  be  sure,  frequently  a  characteristic  dark  brown 
chocolate-colored  appearance;   it  may,   however,    appear 
bright  red  and  arteriaL     Even  the  admixture  of  stomach 
contents  is  not  always  conclusive,  as  vomiting  may  be 
caused  by  severe  hemoptysis.      The  composition  of  the 
clot  may,  however,  be  of  aid.     In  hemoptysis  the  blood 
usually  coagulates  more  quickly  and  more  thoroughly  than 
in  hematemesis,    and  the   clot   shows   on   cross-section 
numerous  pores,  caused  by  the  admixture  of  air,  which 
give  it  a  spongy  appearance.     The  detection  of  sputum 


26     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

flecks  in  the  blood  is  decisive  for  the  diagnosis  of  hemop- 
tysis. In  a  great  number  of  cases,  however,  only  the  his- 
tory and  examination  of  the  patient  can  establish  the 
source  of  hemorrhage. 

2.  Blood-stained  sputum.     The  blood  permeates  in  the 
form  of  flecks  or  streaks,  the  mucoid,  muco-purulent,  or 
purulent  sputum. 

3.  Sputum  thoroughly  mixed  with  blood.     The  ap- 
pearance differs  according  to  the  quality  of  the  blood  con- 
tained. 

(a)  Mucoid;, Bloody  sputum  is  tenacious  and  has  a 
yellow  to  rusty  brown  color.  It  is  characteristic  of  the 
alveolae  and  smallest  bronchi. 

(#)  Serous  bloody  sputum  is  thinly  liquid,  contains 
numerous  air-bubbles  and  has  a  dark  brown  to  black  color. 
It  is  designated  as  prune-juice  expectoration. 

(c)  Purulo-sanguineous  sputum  (that  containing  pus 
and  blood  thoroughly  mixed)  points  to  the  existence  of 
large  cavities,  in  which  it  is  produced  by  the  mixture  of 
purulent  secretion,  with  more  or  less  altered  blood  ingredi- 
ents. Two  forms  of  this  sputum  are  recognized,  depend- 
ing upon  whether  it  is  expectorated  soon  after  its  secretion 
or  after  a  longer  retention  in  the  cavity.  In  the  first  in- 
stance, airless,  nummular  clumps,  with  dirty  red  centres 
and  distinctly  red-stained  periphery,  are  expectorated, 
which  sink  quickly  to  the  bottom  in  water  (ftp.  globosum 
fundum  petens).  In  the  latter  instance,  the  sputum  has 
a  homogeneous  appearance  and  a  dirty  red  to  muddy 
brown  color  (pulmonary  gangrene,  bronchiectasis). 

Especially  Prominent  Ingredients  of  the  Sputum 

The  so-called  kernels  (Corpuscula  oryzoidea)  are  pin- 
head  to  sago- sized  opaque  objects,  yellowish- white  in 
color  and  cheesy  in  consistency.  They  can  be  easily  iso- 


SPUTUM  27 

lated  from  the  purulo-mucoid  sputum  in  which  they  are 
usually  found.  They  have  their  origin  in  cavities,  and 
are  of  diagnostic  worth,  as  they  are  usually  very  rich  in 
tubercle  bacilli  and  elastic  fibres.  These  kernels  should 
not  be  confused  with  tonsillar  plugs  and  pieces  of  food 
which  may  resemble  them.  They  are  distinguished  from 
these  by  .microscopical  examination. 

Dittricli's  plugs  are  grayish-white  particles,  sometimes 
as  large  as  a  bean,  which  are  found  in  the  sediment  of 
sputum  in  pulmonary  gangrene. 

Portions  of  tissue  may  be  present  in  sputum  in  ulcera- 
tive  processes  of  the  respiratory  organs.  They  are  most 


FIG.  2. — Fibrin  Clots  from  Pneumonic  Sputum. 
(After  v.  Jaksch. ) 

frequently  present  in  the  sediment  of  gangrenous  sputum, 
and  appear  as  black  or  dark  gray  villous  shreds,  which, 
when  examined  microscopically,  are  seen  to  be  necrotic 
lung  tissue.  Pieces  of  tumors  may  be  present  in  cases 
of  neoplasm  of  the  lungs;  their  presence  is,  however, 
extremely  rare. 

Curschmann's  spirals  appear  as  spiral  threads  sharply 
defined  against  the  background  of  structureless  sputum. 
They  are  grayish-white  in  color  and  noticeably  firm  in  con- 
sistency (Figs.  3  and  4). 


28     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Fibrin  clots  (Fig.  2)  are  white,  cylindrical,  branch- 
ing structures  which  may  be  several  centimetres  in  length. 
They  result  from  clotting  of  fibrin  in  the  bronchi,  of  which 
they  are  casts.  They  must  not  be  confused  with  the  clots 
of  thickened  mucus  which  macroscopically  resemble  them, 
and  which  are  much  more  common  in  sputum  than  true 
fibrin  clots.  They  can  be  distinguished  from  one  another 
by  microscopical  and  microchemical  examination.  The 
branchings  can  be  more  clearly  seen  after  the  clot  has  been 
washed  in  water. 

Actinomyces  kernels  appear,  principally,  as  compara- 
tively solid  bodies  the  size  of  grains  of  sand  and  yellowish- 
green  or  black  in  color.  There  are  in  addition,  however, 
gray,  easily  crushed  kernels  which  appear  gelatinous  and 
glassy,  resemKLing  clumps  of  mucus. 

Food  particles  are  often  mixed  with  sputum,  and  are 
present  especially  in  the  mucous  portions  coming  from  the 
upper  air-passages. 

Microscopical  Examination 

The  particles  which  are  to  be  examined  microscopically 
are  best  isolated  from  the  surrounding  sputum  by  the  aid 
of  two  platinum  wires,  which  can  be  sterilized  in  a  flame 
before  and  after  using,  smeared  on  a  slide,  covered  with  a 
cover  glass,  and  examined  first  with  the  low,  then  with  the 
high,  power. 

|  In  the  examination  of  fresh  smears,  microchemical  re- 
actions are  often  used.  The  reagents  most  frequently  used 
are  dilute  acetic  acid,  and  an  8  to  10  per  cent,  potassium 
hydrate  solution.  To  obtain  thorough  admixture  of  the 
reagents  with  the  material  to  be  examined,  they  are  rubbed 
together  on  the  slide  before  being  covered  with  the  cover 


The  objects  which  have  attracted  attention  during  the 


SPUTUM  29 

macroscopical  examination  of  the  sputum  over  a  dark  back- 
ground are  the  first  to  be  examined. 

The  Curschmann  Spirals    (Figs.  3,  4),  which,  because 
of  their  tough  consistency,  can  only  be  crushed  with  diffi- 


FIG.  3.— Spirals  from  the  Sputum  (Natural  Size), 
(After  v.  Jaksch.) 


W  1|| 

FIG.  4.— Spirals  from  the  Sputum  (Magnified). 
(After  v.  Jaksch.) 

culty  between  cover  glass  and  slide,  permit  when  held 
against  the  light,  even  macroscopically,  a  distinct  spiral 
form  to  be  seen.  In  the  microscopical  picture  they  pre- 
sent themselves  as  translucent  spirals  composed  of  numer- 
ous closely  placed  and  delicate  convolutions,  in  whose 


30    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

axis  there  usually  runs  a  central  thread.  They  are,  as  a 
rule,  thickly  covered  with  leucocytes,  among  which 
Char  cot- Ley  den  crystals  are  often  present.  Usually  the 
structure  of  the  spiral  becomes  distinct  only  after  the  ad- 
dition of  acetic  acid. 

The  Fibrin  Coagula  are  composed  of  bundles  of  parallel 
refractive  threads,  between  which  more  or  less  numerous 
leucocytes,  as  well  as  erythrocytes,  and  occasionally 
Char  cot- Ley  den  crystals,  are  visible.  The  mucus  coagula, 
•which  resemble  them  microscopically,  are,  however,  com- 
posed of  a  homogeneous  basic  substance,  in  which  leuco- 
cytes are  embedded.  Upon  the  addition  of  acetic  acid 
the  fibrinous  structures  become  clearer,  while  the  mucus 
coagula  become  cloudy,  and,  at  the  same  time,  their  basic 
substance  assumes  a  striated  appearance. 

The  Tissue  Shreds,  which  attract  attention  in  gan- 
grenous sputum,  contain  connective-tissue  fibres  whose 
alveolar  arrangement  identifies  them  as  the  remains  of 
necrotic  lung-tissue.  Elastic-tissue  fibres  are  rarely  recog- 
nizable in  them.  The  connective-tissue  fibres  are  usually 
surrounded  by  a  large  mass  composed  of  various  kinds  of 
bacteria,  fatty  detritus,  fatty  acid  needles,  triple  phos- 
phate crystals,  and  dark  pigment  granules.  The  paren- 
chymatous  shreds,  which  are  present  in  the  sputum  of 
subacute  or  chronic  lung  abscess,, contain  nearly  always, 
on  the  contrary,  elastic  fibres,  either  singly  or  in  alveolar 
arrangement,  and  in  addition  are  composed  of  numerous 
bacteria,  fatty  degenerated  cells,  and  fatty  acid  needles, 
containing  also,  occasionally,  crystals  of  hematoidin  and 
cholesterin  crystals,  which  latter  are  otherwise  rarely  pres- 
ent in  the  sputum. 

Dittrich's  Plugs  consist  principally  of  masses  of  de- 
tritus, and  of  an  exceptional  number  of  different  micro- 
organisms. 


SPUTUM 


31 


Portions  of  the  Echinococcus  (Figs.  5  and  6)  are  expec- 
torated when  echinococci  are  located  in  the  lungs,  or  when 


FIG.  5. — Hyaline  Membrane  of  an  Echinococcus  Cyst. 
(After  v.  Hansemann. ) 

an  echinococcus  cyst  has  ruptured  into  them  from  the 
neighboring  tissues.  In  the  sputum  of  pulmonary  echi- 
nococcus, which  is  always  bloody,  and  may,  through  com- 
munication with  the  liver,  be  bile  or  ochre  colored,  unin- 


j* 


FIG.  6.— Echinococcus  Hooks.     (After  v.  Hansemann.) 

jured  cysts  with  clear  contents  are  occasionally  present; 
in  other  cases  the  characteristic  hooks  may  be  seen,  or 
shreds  of  membrane,  which,  when  finely  teased,  allow  the 


32    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

parallel  striations,  typical  of  echinococcus  membrane,  to 
be  recognized. 

The  hard  yellow  actinomyces  granules  (Figs.  7  and  8) , 
which  attract  attention  during  the  macroscopical  exam- 
ination, appear  under  the  low  power  as  round,  unevenly 
knobby,  finely  granular  objects,  resembling  a  mul- 
berry. When  crushed  under  a  cover  glass,  and  ex- 
amined with  the  high  power,  they  have  a  very  char- 


FiG.  7. — Actinomyces  Granules  (Low  Power). 
(After  v.  Jaksch.) 

acteristic  appearance.  From  a  thick  centre,  composed 
of  a  mass  of  threads,  radiate  numerous  glistening 
fibres,  which  branching  many  times,  end  in  a  bulbous 
enlargement  (actinomyces  clubs).  In  the  central  thready 
mass  are  often  groups  of  spiral,  rod-shaped,  and  coccus- 
like  objects.  The  gray  kernels  resembling  clumps  of 
mucus,  which  are  present  alongside  of  the  characteristic 
actinomyces  clubs,  are  softer  in  consistency  than  these, 
and  are  composed  merely  of  branched  fibrils.  (For  stain- 


SPUTUM  33 

ing,  Gr aril's  method,  with  eosin  as  a  counterstain,  is  suit- 
able, the  fibrils  appearing  bluish-black,  and  the  bulbous 
ends  red.) 

In  mycosis  of  the  lungs  small,  grayish-black  granules 
are  found,  which  consist  of  mold  fungi  (aspergillus  and 
mucor  species) . 

The  unstained  specimen  gives,  in  addition,  informa- 


FIG.  8.— Actinomyces  Granules  (Unstained  Specimen). 
(After  v.  Jaksch. ) 

tion  concerning  the  cellular  elements  of  the  sputum,  and 
the  presence  of  elastic  fibres  and  crystalline  bodies. 

Cellular  Elements  of  the  Sputum 

1.  Epithelial  Cells. — Inasmuch  as  the  sputum  represents 
the  secretion  of  different  portions  of  the  respiratory  tract, 
epithelial  cells  from  every  portion  may  be  present  in  it. 
There  may  be : 

(a)  Large  polygonal  squamous  cells  coming  from  the 
mouth,  pharynx,  or  vocal  cords.  These  squamous  cells 
are  frequently  covered  with  coal  pigment. 

(1))  Cylindrical  cells,  which  are  occasionally  ciliated. 
These  may  come  from  the  pars  respiratoria  of  the  nose, 
from  the  larynx,  or  the  bronchi. 

(c)  Alveolar  E2ntlielial  Cells,—  These,  when  present, 


34    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

are  always  greatly  degenerated,  and  cannot,  as  a  rule,  be 
recognized  with  certainty  as  such.  By  alveolar  epithelial 
cells  are  meant  round,  oval,  or  polygonal,  mono-  or  poly- 
nuclear  cells,  approximately  five  to  six  times  the  size  of  a 
leucocyte.  Their  protoplasm  is  frequently  filled  with 
highly  refractive  fat  drops  or  faintly  glistening  myelin 
drops,  which  may  coalesce  to  larger  drops,  and  then  pro- 
duce the  characteristic  myelin  forms.  Further,  black  coal 
pigment  is  often  seen  in  these  cells  (pigment  cells). 
Under  the  heading  of  alveolar  epithelium,  cells  containing 
reddish-brown  pigment  granules,  the  so-called  heart- failure 
cells,  are  frequently  included.  These  appear  in  great 
numbers  in  the  sputum  in  brown  induration  of  the  lungs. 
This  pigment,  called  hemosiderin,  comes,  as  does  hema- 
toidin,  from  blood  pigment;  but,  in  contrast  to  hematoidin, 
contains  iron. 

Recognition  of  Hemosiderin. — Allow  a  fleck  of  sputum 
containing  heart-failure  cells  to  dry  on  a  cover-glass,  fix 
and  drop  on  it  a  little  2  per  cent,  potassium  ferrocyanide 
solution,  to  which  1  to  8  drops  of  HC1  have  been  added. 
After  half  to  one  hour  the  pigment  granules  will  be  stained 
blue. 

2.  Leucocytes. — These  are  present  in  varying  numbers 
in  every  sputum  and  in  large  quantity,  as  the  principal 
ingredients  of  pus.  They  are  usually  more  or  less  degen- 
erated, being,  as  a  rule,  poly-morpho-nuclear,  and  show- 
ing generally  neutrophilic  granulation.  Numerous  eosino- 
philic  leucocytes  are  found  in  the  sputum  of  asthmatic 
patients.  (Staining  according  to  May  and  Gruenwald, 
cf.  p.  258,  gives  a  good  picture.)  The  leucocytes  con- 
tain frequently,  like  the  so-called  alveolar  epithelium,  pig- 
ment granules,  coal  pigment,  as  well  as  altered  blood  pig- 
ment. It  is  generally  recognized  now  that  the  so-called 
heart-failure  cells  are  not  merely  alveolar  epithelial  cells, 


SPUTUM  35 

but  that,  on  the  contrary,  the  majority  of  them  are  leuco- 
cytes. 

3.  Red  Blood  Corpuscles. — Isolated  red  blood  cells  are 
present  in  every  sputum,  and  have  no  diagnostic  signifi- 
cance. They  point  to  hemorrhage  in  the  respiratory  organs 
only  when  they  are  present  in  great  numbers.  They  may 
be  perfectly  intact  both  in  form  and  color,  or  they  may 
appear  altered,  being  swollen,  or  crenated,  or  having  lost 
their  pigment  (shadow  corpuscles) . 

•Elastic  Fibres— (Fig.  9) 

The  material  to  be  examined  for  the  presence  of  elastic 
fibres  should  be  taken  'from  the  opaque  purulent  portions 


FIG.  9.— Elastic  Fibres  from  the  Sputum.     (After  v.  Jaksch.) 

of  the  sputum.  To  facilitate  the  search  for  elastic  fibres 
the  material  to  be  examined  should  be  mixed  with  a  drop 
of  10  per  cent,  potassium  hydrate  solution,  covered  with 
a  cover  glass,  and  slightly  warmed  over  a  small  flame. 
By  this  means  the  cellular  elements  will  be  decomposed, 
while  the  elastic  fibres  remain  unchanged.  When  the 
search  made  in  this  manner  is  unsuccessful,  several  clumps 


36     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

of  sputum  should  be  put  in  a  test-tube,  an  equal  quantity 
of  10  per  cent,  potassium-hydrate  solution  added,  and  the 
mixture  boiled  until  it  appears  homogeneous ;  then  diluted 
with  four  times  as  much  water  and  centrifuged.  Smears 
should  be  made  from  the  sediment  and  examined  with  a 
magnification  of  about  300. 

The  elastic  fibres  appear  highly  refractive,  characteris- , 
tically  wavy,  clear  cut,  double  contoured,  and  frequently 


FIG.  10.— Leyden  Crystals  (Magnified  300  Times). 

branching.  They  lie  singly,  in  bundles  of  longitudinal 
fibres,  or  have  a  net  or  mesh  like  (alveolar)  arrangement. 
It  must  be  remembered  that  elastic  fibres  coming  from 
food  may  be  present  in  the  sputum.  Only  when  elastic 
fibres  in  alveolar  arrangement  are  found  can  it  be  said  with 
certainty  that  they  come  from  the  lungs. 

Crystalline  Bodies 

Of  the  crystalline  bodies,  Char  cot- Ley  den  crystals  have 
particular  significance  (Fig.  10).     These  appear  as  clear, 


SPUTUM  37 

glistening,  pointed,  octahedra,  resembling spermin crystals 
in  appearance.  They  are  present  in  particularly  large 
numbers  when  the  sputum  has  stood  for  a  time  exposed  to 
the  air.  Their  formation  may  be  observed  microscopically 
in  fresh  cover-glass  specimens. 

In  addition,  fatty  acid  needles  may  be  present,  which 
can  be  distinguished  from  objects  resembling  them — for 
example,  elastic  fibres,  by  the  fact  that,  when  the  slide  is 
carefully  warmed,  they  melt  and  coalesce,  forming  fat 
drops.  Further,  crystals  of  calcium  oxalate,  triple  phos- 
phate, and  cholesterin,  as  well  as  of  leucin  and  tyrosin, 
may  be  seen;  and,  finally,  hematoidin  crystals  may  be 
present  in  the  form  of  reddish-yellow  or  ruby-red  rhomboid 
plates,  and  wavy  needles  which  lie  free  or  radiate  in  tufts 
from  the  plates. 

BACTERIOLOGICAL   EXAMINATION   OF  THE   SPUTUM 

Preparation  of  the  Sputum  for  Examination 

Only  such  sputum  is  suitable  for  bacteriological  exam- 
ination as  surely  comes  from  the  diseased  air-passages. 
For  examination  the  true  bronchial  or  lung  sputum  must 
be  separated  from  the  secretions  which  have  become  mixed 
with  it  during  its  passage  through  the  upper  respiratory- 
tract,  as  the  latter  frequently  contain  exactly  the  bacteria 
which  are  significant  of  pulmonary  diseases. 

The  Pfeiffer  and  Kocli-Kitasato  methods  serve  this 
purpose.  According  to  Pfeiffer' s  suggestion  the  sputum 
is  poured  into  a  sterile  Petri  dish,  which  is  placed  over  a 
dark  background,  and  a  thick  opaque  portion  is  taken 
from  it  and  placed  upon  the  cover  of  the  dish.  This  por- 
tion is  thoroughly  spread  out  by  the  aid  of  two  platinum 
wires,  and  a  distinctly  purulent  particle  (the  nucleus)  is 
isolated. 


38    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

According  to  the  Koch-Kitasato  method  a  sputum  ball 
is  washed  in  a  series  of  dishes  filled  with  sterile  water, 
which  is  thoroughly  stirred  with  a  heavy  platinum  needle. 
In  this  manner  the  sputum  ball  quickly  becomes  smaller, 
and  finally  separates  into  minute  particles,  from  which  a 
small  pus  kernel  is  taken  for  examination.  Czaplewski 
has  modified  this  method  by  shaking  the  sputum  ball  in 
three  successive  tubes  of  peptone  water. 

The  fleck,  having  in  one  way  or  another  been  freed 
from  the  adherent  mucus  and  bacteria,  which  it  has  col- 
lected in  its  passage  through  the  upper  respiratory  tract, 
may  now  be  used  for  the  preparation  of  smears,  for  plant- 
ing cultures,  or  for  animal  inoculation. 

I.  Examination  of  Stained  Smears. — With  the  aid  of  a 
platinum  wire  the  fleck  is  carefully  smeared  on  cover 
glasses  held  in  cornet  forceps,  either  at  once  or  when  the 
sputum  contains  much  fibrin,  after  the  addition  of  a  drop 
of  sterile  water.  After  the  smears  have  been  dried  in  the 
air,  and  have  been  fixed  by  passing  three  times  -through  the 
flame,  they  are  stained  as  follows : 

1.  According   to   one   of    the   methods    for   staining 
tubercle  bacilli. (cf.  p.  329). 

2.  With   carbol   fuchsin.       Dilute  carbol    fuchsin  is 
dropped  on  the  smear,  heated  to  the  steaming-point  over 
a  small  flame,  and  at  once  washed  off,  as  the  details  are 
hard  to  recognize  in  too  deeply  stained  specimens. 

3.  According  to   Gram  (cf.  p.  330).     The  first  men- 
tioned stain  serves  merely  for  the  detection  of  tubercle 
bacilli.     The  smear,  stained  with  dilute  carbol  fuchsin, 
gives  information  concerning — 

(#)  The  origin  of  the  sputum. 

(b)  The  bacteria  present  other  than  tubercle  bacilli. 

(c)  The  value  of  the  bacteriological  findings. 

It  is  not  always  possible  to  determine  the  origin  of  the 


SPUTUM  39 

sputum  with  certainty  from  the  microscopical  picture, 
although  the  epithelial  cells  present  in  the  sputum,  which 
have  been  called  by  Czaplewski  "guide  cells,"  are  of  as- 
sistance. 

When  the  secretion  comes  from  the  mouth,  pharynx, 
or  nasopharynx,  numerous  large  squamous  cells,  which 
are  usually  .thick  with  bacteria,  are  seen  in  the  smear,  and 
in  addition  more  or  less  numerous  pus  cells,  depending 
upon  the  stage  of  the  inflammation. 

Nasal  secretion  which  has  been  aspirated  and  coughed 
out  shows,  besides  a  varying  quantity  of  leucocytes,  cylin- 
drical cells  which  are  occasionally  ciliated,  as  well  as 
squamous  cells  from  the  pharyngeal  sputum,  which  is 
usually  mixed  with  it.  Further,  the  rich  bacterial  flora 
of  the  nose  and  pharynx  is  always  represented  by  a  great 
number  of  different  micro-organisms. 

Bronchial  and  pulmonary  sputum  contains,  besides  pus 
cells,  cylindrical  epithelial  cells  and  the  pigment  cells, 
which  are  especially  characteristic  of  it. 

Inasmuch  as  nearly  all  the  micro-organisms  which  come 
into  question  in  sputum  examination,  with  the  exception 
of  tubercle  bacilli,  are  easily  stained  with  dilute  aniline 
dyes,  they  can  be  clearly  seen  in  the  specimens  stained 
with  dilute  carbol  fuchsin. 

When  a  certain  kind  of  bacterium  is  found  by  repeated 
examination  in  large  numbers  in  sputum  coming  from  the 
deeper  air-passages,  it  is  proper  to  assume  that  these  bac- 
teria have  an  etiological  connection  with  the  disease. 
When,  on  the  contrary,  the  specimen  shows  a  mixture  of 
different  kinds  of  bacteria,  the  findings  have  no  diagnostic 
value,  unless  it  is  determined  that  these  were  present  be- 
fore the  expectoration  of  the  sputum,,  and  are  not  due  to 
subsequent  contamination. 

The  specimen  stained  by  Gram's  method  aids  in  find' 


40     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

ing  6rraw-positive  bacteria,  which,  because  of  their  dark 
staining,  are  prominent  against  the  brown  background  of 
decolorized  cellular  elements. 

Further,  it  shows  how  the  bacteria  seen  in  the  carbol- 
fuchsin  specimen  act  toward  the  Gram  method,  and  thus 
aids  in  identifying  them. 

II.  For  cultural  examination  plain  agar,  glycerine  agar, 
blood  agar,  blood  serum,  and  bouillon  are,  as  a  rule,  used; 
other  culture  media,  as  gelatine,  potato,  etc. ,  are  but  ex- 
ceptionally used.      The  washed  fleck  is  smeared   either 
immediately  or  after  being  floated  in  physiological  salt 
solution.     When  the  presence  of  many  germs  capable  of 
development  is  suspected,  the  same  fleck  is  smeared  on  a 
number  of  culture  media  to  obtain  isolated  colonies. 

III.  Animal   Inoculation. — White   mice,     guinea-pigs, 
and  rabbits  are  the  test  animals  most  frequently  used  in 
sputum  examination.     The  washed  sputum  fleck  is  either 
introduced  directly  into  a  pocket  under  the  skin,  or,  after 
being  floated  in  a  sterile  0.85  per  cent,  sodium  chloride 
solution,  is  injected  subcutaneously  or  intraperitoneally. 

Detection  of  Tubercle  Bacilli.  (Plate  II,  Fig.  C.)- 
Material  for  smears  should  always  be  taken  from  a  num- 
ber of  suspected  places  in  the  sputum,  the  so-called  ker- 
nels being  especially  sought  after.  In  specimens  prepared 
after  the  method  described  on  p.  330,  the  tubercle  bacilli 
are  stained  red,  while  the  other  bacteria  and  cellular  ele- 
ments are  blue.  The  tubercle  bacilli  present  themselves 
as  slim  rods  of  varying  length,  and  do  not  always  appear 
perfectly  straight,  but  are  often  slightly  curved.  They  lie 
in  groups,  singly,  or  in  pairs,  which  may  be  parallel  or  at 
right  angles  to  one  another.  They  are  often  of  uneven 
thickness,  or  irregularly  granular.  Colorless  spots  are 
seen  between  the  stained  granules,  so  that  the  bacilli 
resemble  a  string  of  pearls.  Further  single,  small,  blue 


SPUTUM  41 

to  blackish  red  "venous"  stained  objects  are  found,  which 
are  thought  to  be  fragments  of  bacilli,  and  which  Spengler 
has  called  "splinters."  Fibril  forms,  with  true  branch- 
ing and  bulbous  ends,  have  been  very  rarely  observed  in 
sputum. 

The  number  of  bacilli  found  in  a  smear  gives  no  clue 
to  the  course  of  the  disease,  for  their  number  is  most  vari- 
able, both  in  different  portions  of  the  same  sputum,  and 
in  sputum  expectorated  at  different  times  of  the  day. 
Nevertheless,  the  question  of  counting  tubercle  bacilli  may 
arise,  in  which  case  the  method  of  Gaffky  or  that  of 
CzaplewsJci  may  be  used. 

GAFFKY 's  SCALE 

1  =  1 — 4  bacilli  in  an  entire  specimen. 

2  =  on  the  average  only  1  bacillus  in  a  number  of  fields. 
3=         "  "  1  bacillus  to  a  field. 

4 =  "  "  2—3  bacilli  to  a  field. 

5=  «  «  4—6      "       "       " 

6=  "  "  7 12     "       "       " 

7—  "  quite  a  few  bacilli  to  a  field. 

8=  "  "  numerous         "         "       " 

9=  very  numerous  bacilli  to  a  field. 
10  =  a  very  large  number  of  bacilli  in  every  field. 

CZAPLEW SKI'S  METHOD 

1 '  In  the  numerator  of  a  fraction  is  written  the  number 
of  bacilli  which  are  counted  in  a  field;  in  the  denomi- 
nator, the  number  of  fields  counted.  If  in  one  or  more 
entire  smears  only  a  few  bacilli  are  counted,  then  the 
denominator  is  written  as  a  Roman  numeral,  which 
represents  the  number  of  smears  examined.  For  ex- 
ample : 


42     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

/» 
=6  bacilli  in  a  field. 

-y-=  innumerable  bacilli  in  a  field. 

—  =1  bacillus  in  five  fields. 
5 

2 
— =  2  bacilli  in  an  entire  smear. 

—  =1  bacillus  in  six  entire  smears, 

and  so  forth.     It  is  perhaps  well  to  express  the  minimum 
and  maximum  number  observed  I  for  example  -  — ,  etc.  I 

and  also  the  average  found  in  a  number  of  fields 

/,  0—6      3  \ 

I  for  example   — —  =— -  I 

"It  should,  however,  be  mentioned  that  the  diameter 
of  a  field  varies  with  change  of  objective,  ocular,  or  tube 
length,  and,  therefore,  the  results  are  of  value  only  when 
arrived  at  with  the  same  optical  combination. ' ' 

Sedimentation. — This  procedure,  first  used  by  Biedert, 
has  for  its  object,  by  liquefying  the  sputum  and  rendering 
it  homogeneous,  the  collection  in  the  sediment  of  the  iso- 
lated and  scattered  bacilli,  that  they  may  be  more  easily 
detected.  A  great  number  of  sedimentation  methods  have 
been  proposed. 

Beitzke,  who  has  tested  the  various  methods,  recom- 
mends especially  the  Muelilhaeuser-Czaplewski  method, 
which  is  carried  out  in  the  following  manner :  * 

"The  sputum  is  placed  in  a  cylindrical  glass,  about 
four  times  as  much  0.2  per  cent,  sodium  hydrate  solution 

1  "  Hygienische  Rundschau,"  12,  No.  1. 


SPUTUM  43 

added,  the  glass  closed  with  a  rubber  cork  and  shaken 
vigorously  for  a  minute.  This  is  often  sufficient  to  pro- 
duce an  even,  thinly  liquid,  and  no  longer  mucoid,  sub- 
stance, in  which  no  large  flecks  are  visible.  When  this  is 
not  sufficient,  more  alkali  is  added,  the  glass  again  vigor- 
ously shaken,  and  so  on,  ifttil  such  a  fluid  is  produced. 
As  a  rule,  not  more  than  eight  times  as  much  alkali  as 
sputum  will  be  needed,  though  I  have  occasionally  found 
it  necessary  to  dilute  twelve  times.  When  the  sputum 
has  in  this  way  wholly  lost  its  mucoid  aspect,  and  has 
become  entirely  liquid,  it  is  poured  into  a  porcelain  or 
enamel  dish,  and  under  constant  stirring  heated  to  the 
boiling-point. 

"When  the  sputum  has  become  entirely  homogeneous, 
one  or  two  drops  of  phenol-phthalein  solution  are  added, 
then,  drop  by  drop,  under  vigorous  stirring,  5  per  cent, 
acetic  acid  solution,  until  the  red  color  disappears.  If 
the  mixture  is  not  stirred  energetically  enough,  it  is  easy 
to  add  too  much  acetic  acid,  in  which  case  the  whole  pur- 
pose of  the  procedure  will  be  thwarted  by  the  heavy  pre- 
cipitation of  mucin.  This  is  sure  to  be  the  case  when  the 
liquid  had  even  the  slightest  mucoid  character  before  its 
neutralization.  After  its  neutralization  the  liquefied 
sputum  is  diluted  with  water,  or  with  two  parts  alcohol, 
and  either  allowed  to  stand  or  is  centrifugalized. "  It  is 
possible  to  centrifugalize  the  entire  quantity  in  a  single 
centrifuge  tube  by  continually  pouring  off  the  fluid  from 
the  sediment  and  filling  the  tube  anew,  until  the  entire 
sputum  has  been  used.  From  the  sediment  obtained  in 
this  manner  smears  are  made  and  stained. 

The  cultivation  of  tubercle  bacilli  from  the  sputum  hardly 
enters  into  consideration  for  diagnostic  purposes,  as  it  is 
too  tedious  and  frequently  fails.  Kitasato  first  succeeded 
according  to  the  following  method:  The  patient  is  re- 


44     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

quested  to  thoroughly  cleanse  his  mouth  with  a  gargle, 
and  then  to  expectorate  into  a  sterile  dish.  A  sputum 
ball  is  washed  in  the  manner  described  above,  torn  apart 
under  water  in  the  last  dish,  a  particle  taken  from  its 
centre  and  examined  in  a  stained  smear.  If  the  sputum 
contains  tubercle  bacilli  in  pure  culture,  glycerine  agar 
and  blood  serum  tubes  are  inoculated  with  a  second  par- 
ticle. The  cotton  plugs  are  cut  short  and  pressed  into  the 
tubes,  and  in  order  to  prevent  drying  of  the  culture  media 
the  tube  is  sealed  with  a  rubber  cap,  still  moist  from  the 
sublimate  solution  in  which  it  has  been  disinfected. 
After  two  weeks'  growth  the  colonies  are  visible  as  moist, 
glistening,  smooth,  round  flecks. 

W.  Hesse l  recommends  the  following  procedure  for  the 
cultivation  of  tubercle  bacilli  from  sputum.  The  sputum 
raised  from  the  depth  of  the  lungs,  when  possible  without 
the  addition  of  saliva,  is  expectorated  into  a  sterile  Petri 
dish.  A  portion  of  the  sputum  the  size  of  a  pea  is  placed 
on  the  culture  media  (plate  culture)  and  divided  into 
twenty  to  thirty  small  flecks. 

Preparation  of  tlie  Culture  Media. — One  part  agar- 
agar,  3  parts  glycerine,  96  parts  water,  are  mixed,  filtered, 
and  placed  in  test-tubes  of  a  capacity  of  50  cc,  which 
must  be  of  resistant  glass  containing  no  alkali,  so  that 
each  tube  contains  20  cc.  The  tubes  containing  the  cul- 
ture media  are  steam  sterilized  for  about  three  hours. 
The  culture  media  must  have  the  same  alkalinity  as  the 
sputum  which  is  to  be  examined.  For  this  purpose  six 
tubes  are  taken;  to  five  of  them*  is  added  decinormal 
KOH  in  quantities  of  0.2,  0.5,  1.0,  2.0,  and  5.0,  from 
which  six  plates  are  made  and  inoculated  in  the  above 
manner.  It  is  then  to  be  expected  that  one  of  the  plates 

1  Centralblatt  f.  Bakt.,  xxxv.  No.  3. 


SPUTUM  45 

will  have  the  desired  alkalinity.  Or  the  following  method 
is  employed :  To  five  of  six  test-tubes  containing  20  cc  of 
water,  decinormal  KOH  is  added  in  quantities  of  0.2, 
0.5,  1.0,  2.0,  and  5.0,  and  a  drop  from  each  is  placed  on 
a  strip  of  litmus  paper.  One  of  the  6  drops  will  color  the 
paper,  approximately  as  blue  as  does  the  sputum,  and 
will  show  how  much  alkali  should  be  added  to  the  media 
to  give  it  practically  the  same  reaction  as  the  sputum. 

A  strip  of  asbestos  is  placed  between  the  cover  and  bot- 
tom of  the  inoculated  dishes;  they  are  held  tightly  closed 
by  a  rubber  band,  and  placed  with  the  culture  media  up- 
permost in  an  incubator.  After  one  to  two  days  the  plates 
are  tested  by  means  of  ./TMsc/i-preparata  made  in  the  fol- 
lowing manner :  a  sterile  cover  glass  resting  over  the  open- 
ing of  a  test-tube  is  carefully  pressed  upward  against  the 
surface  of  the  culture  where  there  is  a  sputum  fleck.  The 
cover  glass  is  then  lifted  from  the  culture  media  with  a 
platinum  wire  and  grasped  with  forceps.  The  growth  of 
the  tubercle  bacilli  can  be  detected  in  the  A7a^c7^-prepa- 
rata.  The  colonies  may  be  distinctly  seen  after  a  few 
days'  growth  with  the  low  power,  and  after  a  few  weeks 
with  the  naked  eye. 

More  reliable  results  are  to  be  obtained  from  animal 
inoculation  than  from  cultural  methods.  Half-grown 
guinea-pigs,  weighing  about  250  grammes,  are  used,  as 
they  are  more  sensitive  than  older  animals.  They  are  in- 
oculated subcutaneously,  'either  on  the  abdomen  or  on  the 
thigh,  by  introducing  a  washed  fleck  into  a  niche  under 
the  skin,  or  by  injecting  the  fleck  suspended  in  physio- 
logical salt  solution.  The  inoculation  must  be  made  in 
an  aseptic  manner,  after  the  skin  has  been  shaved  and 
washed  with  alcohol.  The  subcutaneous  inoculation  has 
the  advantage  over  the  intraperitoneal  that  it  enables  one 
to  follow,  step  by  step,  the  advance  of  the  tuberculosis. 


46    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

The  tubercular  process  produces  at  first  an  infiltration  at 
the  site  of  inoculation,  next  attacks  the  regional  lymph- 
nodes,  and  then  spreads  to  the  viscera. 

After  three  to  four  weeks  tubercle  bacilli  may  be  de- 
tected in  the  pus  of  an  extirpated  node,  or  in  the  fistula 
at  the  site  of  injection.  If  the  animal  is  killed  four  to 
six  weeks  after  inoculation,  the  autopsy  reveals  the  follow- 
ing conditions:  infiltration  at  the  site  of  injection,  the 
neighboring  lymph-nodes  enlarged  and  caseated,  the  in- 
ternal lymph-nodes  enlarged,  and  more  or  less  numerous 
tubercles  in  the  viscera,  especially  in  the  spleen  and  liver. 
The  search  for  tubercle  bacilli  must  never  be  omitted. 
For  this  purpose  a  tubercle  is  placed  with  a  scalpel  or 
platinum  wire  on  a  slide,  crushed  and  smeared  over  it, 
and  stained  in  the  usual  manner.  As  a  rule  only  a  few 
isolated  bacilli  are  found  in  the  tubercles.  It  is  easier  to 
obtain  cultures  of  tubercle  bacilli  from  tubercles  than  from 
sputum.  For  this  purpose  a  series  of  blood-serum  tubes 
are  inoculated  and  closed  with  sterile  rubber  caps.  The 
material  to  be  inoculated  must  be  crushed  between  two 
sterile  slides  or  scalpels,  and  thoroughly  mixed  with  the 
culture  media.  After  two  weeks  at  the  earliest,  dry,  yel- 
lowish-white flakes  appear,  from  which  finally  a  pure  cul- 
ture develops,  forming  a  closely  coherent,  wrinkled  mem- 
brane. 

Differential  Diagnosis. — Although  acid-fast  bacilli  other 
than  tubercle  bacilli  may  appear  in  sputum,  their  presence 
is  so  very  rare  that  this  does  not  detract  from  the  diag- 
nostic value  of  stained  smears.  They  have,  however,  been 
observed  in  cases  of  pulmonary  gangrene,  bronchiectasis, 
and  putrid  bronchitis.  Therefore,  particular  care  should 
be  taken  in  making  a  diagnosis  when  such  conditions  are- 
present.  These  acid-fast  bacilli  may,  to  be  sure,  differ  in 
their  form  from  tubercle  bacilli.  They  are  usually  slim- 


SPUTUM  47 

mer  and  straighter  than  tubercle  bacilli  and  slightly 
pointed  at  the  ends,  but  these  differences,  when  compared 
with  the  varying  appearance  of  tubercle  bacilli,  are  too 
slight  to  allow  a  certain  diagnosis  to  be  made.  Although 
the  other  acid-fast  bacilli  are  frequently  more  easily  de- 
colorized by  absolute  alcohol  than  tubercle  bacilli,  this  is 
by  no  means  a  constant  characteristic,  and  should  not  be 
relied  upon  in  making  a  differential  diagnosis.  In  cul- 
tures they  differ  from  tubercle  bacilli  in  that  they  develop 
more  quickly,  and  at  room  temperature  on  artificial  cul- 
ture media.  After  twenty- four  to  forty-eight  hours' 
growth  on  glycerine  agar,  white,  glistening  colonies  the 
size  of  a  pin's  head  have  appeared,  which  gradually  co- 
alesce, forming  a  white,  creamy  coating.  After  longer 
growth,  the  lustre  disappears  and  the  surface  looks  dry. 
At  room  temperature  an  orange-yellow  pigment  gradually 
forms.  Animal  inoculation  presents  the  surest  means  of 
differentiation,  as  the  other  acid-fast  bacilli  never  produce 
the  typical  appearance  of  tuberculosis.  To  be  sure,  when 
injected  with  butter  into  the  peritoneal  cavity  of  guinea- 
pigs,  they  excite,  in  addition  to  a  fibrinous  peritonitis, 
changes  which  resemble,  macroscopically,  tubercular  nod- 
ules (true  tubercles)  but  which,  when  examined  histologi- 
cally,  differ  widely  from  them,  in  that  they  show  a  more 
exudative  than  productive  character,  and,  further,  that 
Langhans*  giant  cells,  as  well  as  epithelioid  cell  nests,  are 
lacking.  Finally,  in  contrast  to  the  true  tubercular  nod- 
ules they  contain  usually  numerous  acid-fast  bacilli. 

Further,  lepra  bacilli,  which  are  also  acid  fast,  must 
be  considered  in  making  a  diagnosis.  They  differ,  how- 
ever, from  tubercle  bacilli,  in  that  they  stain  easily  with 
watery  gentian  violet  and  fuchsin  solutions  (cf .  p.  832) . 
They  are  rarely  seen  singly,  but  lie  usually  within  the 
cells  arranged  in  a  group  resembling  a  bunch  of  cigars. 


48     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

In  this  case,  also,  the  final  decision  depends  upon  the  re- 
sults of  cultural  tests  and  animal  inoculation.  The  nega- 
tive result  of  both  is  evidence  in  favor  of  leprosy,  as,  up 
to  the  present  time,  attempts  to  cultivate  lepra  bacilli,  or 
to  infect  animals  with  them,  have  failed. 

The  other  bacteria  which  appear  in  the  sputum,  both 
ae  independent  exciters  of  disease  and  as  producers  of 
mixed  infection  in  tuberculosis,  are  seen  in  the  specimens 
stained  with  dilute  carbol  fuchsin,  and  according  to 
Gram. 

Pneumococci  (Plate  II,  Fig.  D,  and  Plate  III,  Fig.  E) 

Microscopical  Examination. — Pneumococci  present 
themselves,  as  a  rule,  as  diplococci,  which  are  pointed  on 
one  side,  usually  the  outer,  less  frequently  the  inner,  while 
the  opposite  side  appears  rounded  (lancet  form) .  They 
have  a  distinct  capsule,  and  stain  according  to  Gram. 
They  frequently  form  short  chains.  The  picture  which 
the  stained  smears  present  is  often  so  characteristic,  that 
from  the  microscopical  specimen  alone  the  diagnosis 
"pneumococci"  can  be  made.  In  other  cases,  however, 
cultures  and  animal  inoculation  must  be  used  for  their 
identification. 

Cultivation. — On  glycerine  agar  pneumococci  develop 
small  colonies  resembling  dew-drops;  in  bouillon  they 
grow  frequently  in  long  chains.  The  capsules  fail  to  ap- 
pear, as  a  rule,  in  the  growths  on  artificial  culture  media. 
They  are,  however,  not  infrequently  found  in  specimens 
made  from  blood-serum  cultures. 

Animal  Inoculation. — The  most  suitable  test  animals 
are  rabbits  and  white  mice.  The  sputum  fleck  is  dissolved 
in  physiological  salt  solution  and  injected  subcutaneously, 
about  0.1  cc  for  a  mouse,  and  about  0.5  to  1  cc  for  a  rab- 


SPUTUM  49 

bit.  After  twenty-four  to  forty-eight  hours  the  animals 
die  of  pneumococcus  septicemia.  In  the  blood  from  the 
heart  and  in  the  viscera,  innumerable  pneumococci  with 
capsules  are  found.  The  easiest  method  of  isolating  pneu- 
mococci from  the  sputum  is  animal  inoculation. 

Streptococci 

Microscopical  Examination.— Streptococci  are  arranged 
in  chains  of  varying  length,  whose  individual  components 
are  spherical.  Diplo  forms,  which  are  found  to  be  strep- 
tococci only  after  cultivation,  are  frequently  present  in 
the  sputum.  They  stain  according  to  Gram. 

Cultivation.—  On  agar  they  grow  rather  slowly  in  very 
small,  delicate,  transparent  colonies.  With  the  low  power, 
the  centre  appears  finely  granular  and  darker  than  the 
periphery;  the  latter  is  either  regular  and  smooth,  or  may 
be  irregular  and  frayed,  allowing  the  individual  chains  of 
streptococci  to  be  seen.  In  smears  taken  from  agar  cul- 
tures, the  chain  formation  is  frequently  absent.  In  bouil- 
lon they  produce,  as  a  rule,  a  flaky  precipitate  without 
rendering  it  cloudy,  and  develop  long  chains  (Strepto- 
coccus lone/us) ;  in  rarer  cases  they  render  the  bouillon 
cloudy,  and  form  shorter  chains  (Streptococcus  Irevis). 
They  do  not  liquefy  gelatine. 

The  Streptococcus  mucosus  is  surrounded  by  a  distinct 
capsule  and  is  characterized  by  its  colonies. 

For  the  differential  diagnosis  between  streptococci  and 
pneumococci  ox-gall  is  used;  0.5  cc  of  ox-gall  are  added 
to  2  cc  of  the  bouillon  culture.  If  pneumococci  or  strep- 
tococci are  present,  the  cloudy  mixture  will  clear  up  after 
a  few  minutes,  while  the  other  streptococcus  species  will 
not  change  the  appearance  of  the  gall,  because  of  the  bac- 
teriolytic  action  of  the  gall  toward  the  pneumococci  and 
the  streptococcus  mucosus. 


50    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Animal  inoculation  is  not  necessary  in  making  a  diag- 
nosis. 

Staphylococci 

The  Staphylococcus  aureus,  or  albus,  is  usually  pres- 
ent in  the  sputum,  more  rarely  the  Stapliylococcus 
citreus. 

Microscopical  Examination. — Staphylococci  appear  as 
round  cocci,  usually  arranged  like  a  bunch  of  grapes,  and 
stain  according  to  Gram. 

Cultivation. — On  agar  they  produce  large,  round, 
slightly  elevated,  non-transparent  colonies  of  yellow 
(Stapliylococcus  aureus),  white  (Stapliylococcus  albus),  or 
lemon-yellow  (Stapliylococcus  citreus)  color.  All  true 
Staphylococci  liquefy  gelatine  and  render  bouillon  cloudy. 
Animal  inoculation  is  unnecessary  in  making  a  diagnosis. 

Micrococcus  Tetragenus 

The  Micrococcus  tetragenus  appears  in  the  sputum  only 
as  producer  of  mixed  infection  in  tuberculosis. 

Microscopical  Examination. — The  cocci  are  round  or 
oval,  of  varying  size,  and  lie  in  tetrads  within  a  capsule. 
They  stain  according  to  Gram. 

Cultivation. — On  agar  they  produce  white,  non-trans- 
parent, moist,  glistening  colonies,  at  the  periphery  of 
which,  when  examined  with  the  low  power,  the  tetrad 
arrangement  can  be  seen.  On  gelatine  plates  they  appear, 
at  first,  as  small  white  points,  which  soon  increase  in  size 
and  cover  the  gelatine  with  a  glistening,  porcelain-like 
coating.  Bouillon  remains  clear,  though  a  moderate  pre- 
cipitate forms. 

Animal  Inoculation. — White  mice  are  particularly  sen- 
sitive, and  die  of  septicemia  within  a  few  days  after  the 
infection. 


SPUTUM  51 

Micrococcus  Catarrhalis  (Plate  III,  Fig.  F) 

The  Micrococcus  catarrhalis  appears  in  the  sputum  as 
the  exciting  cause  of  bronchitis  and  broncho-pneumonia, 
particularly  in  children,  but  also  in  adults,  either  alone 
or  together  with  other  exciters  of  inflammation,  especially 
streptococci  and  influenza  bacilli. 

Microscopical  Examination.— \.i  appears  as  a  diplococ- 
cus,  or  a  tetracoccus,  but  never  forms  chains.  It  resem- 
bles the  gonococcus  very  closely,  both  in  form  and  posi- 
tion; it  is,  however,  much  larger.  In  the  acute  stage  the 
cocci  lie  frequently  outside  the  cells,  but  later  often  with- 
in the  leucocytes,  closely  grouped  around  the  nucleus. 
Like  the  gonococcus,  it  is  decolorized  by  Gram. 

Cultivation. — It  grows  on  neutral  or  slightly  alkaline 
agar,  but  more  luxuriantly  on  blood  agar  and  serum  agar. 
After  twenty- four  hours'  growth  it  produces  slightly  ele- 
vated, grayish-white,  glistening  colonies,  having  the 
crumbling,  gritty  consistency  of  mortar.  Examined  with 
the  low  power,  they  are  yellowish-brown  in  color,  unevenly 
granular,  and  have  a  very  irregular,  ragged  outline. 
Gelatine  is  not  liquefied.  It  produces  a  precipitate  in 
bouillon  without  clouding  it,  and  after  some  days  a  scum 
appears  on  its  surface. 

Influenza  Bacillus  (Plate  IV,  Fig.  G) 

Microscopical  Examination. — Influenza  bacilli  are  very 
small,  ovoid  rods,  which  are  decolorized  by  Gram.  They 
lie  frequently  within  the  cells,  and  appear  usually  in  great 
quantities  in  the  sputum,  so  that  the  smear  looks  as  if  it 
had  been  powdered  with  them.  In  stained  sputum  smears 
they  resemble  closely  the  Bacillus  pyocyaneus,  from  which, 
however,  they  are  easily  distinguished  by  cultural  methods 
(cf.  B.  pyocyaneus) 


52    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Cultivation. — Influenza  bacilli  do  not  develop  on  plain 
agar.  They  grow  best  on  blood  agar  and  in  blood  bouil- 
lon. On  the  former  they  produce  clear  colonies,  resem- 
bling dew-drops,  which  show  no  tendency  to  coalesce. 
When  closely  crowded,  they  run  together,  forming  larger 
drops,  though  even  then  the  individual  colonies  may  be 
distinguished.  In  blood  bouillon  they  produce  delicate 
white  flakes. 

In  cultural  examination  of  the  sputum,  besides  blood 
agar,  plain  agar  is,  as  a  control,  inoculated  with  bouil- 
lon in  which  the  material  for  examination  is  suspended. 
When  influenza  bacilli  are  present,  there  must  be  no 
growth  on  the  plain  agar,  while  on  the  blood  agar  the 
above  described  colonies  appear. 

Animal  Inoculation. — Influenza  bacilli  do  not  infect 
the  usual  test  animals. 

Streptobacilli  have  been  found  as  producers  of  mixed 
infection  in  tuberculosis.  They  belong  to  the  group  of 
influenza  bacilli,  and  have  the  same  cultural  character- 
istics, but  differ  morphologically,  in  that  they  are  con- 
siderably larger,  and  have  a  surrounding  capsule. 

Diplobacillus  of  Friedlaender  (Pneumobacillus) 

Microscopical  Examination. — The  pneumobacilli  are 
plump  rods  with  rounded  ends,  varying  greatly  in  size  and 
form,  often  resembling  cocci.  They  lie  in  pairs,  and  pos- 
sess usually  a  distinct  capsule,  which  is  especially  con- 
spicuous in  sputum  smears,  in  contrast  to  those  made 
from  cultures.  They  are  decolorized  by  Gram. 

Cultivation. — They  grow  at  room  or  incubator  tem- 
perature upon  the  usual  culture  media,  and  produce  either 
grayish-white,  moist,  glistening  and  slimy,  or  firmer,  non- 
transparent  colonies.  They  do  not  liquefy  gelatine,  but 


SPUTUM  53 

frequently,  after  longer  growth,   stain   it   brown.     They 
ferment  grape  sugar,  but  do  not  coagulate  milk. 

Animal  Inoculation. — White  mice  die  within  twenty- 
four  to  forty-eight  hours  after  subcutaneous  or  intra- 
peritoneal  inoculation.  Numerous  diplobacilli  having 
capsules  are  found  in  the  blood  and  viscera. 

Bacillus  Pyocyaneus 

The  Bacillus  pyocyaneus  has  been  reported  as  a  pro- 
ducer of  mixed  infection  in  tuberculosis.  The  sputum  is 
stained  by  its  pigment  blue  or  bluish-green,  and  has  a 
characteristic  aromatic  odor. 

Microscopical  Examination. — The  bacilli  appear  as 
small,  slim  rods,  which  are  decolorized  by  Gram. 

Cultivation. — On  culture  media  B.  pyocyaneus  pro- 
duces a  pigment  which  stains  the  entire  media.  On  agar 
its  colonies  are  round,  with  a  smooth  circumference;  on 
gelatine  they  are  flat,  with  an  irregular  border,  and  are 
soon  surrounded  by  a  liquefied  area.  Bouillon  is  markedly 
clouded,  milk  coagulated  and  peptonized.  The  B.  pyo- 
cyaneus differs  from  the  influenza  bacillus  in  that  it  is 
easily  cultivated  on  the  usual  culture  media,  produces 
pigment,  and  is  motile. 

Animal  Inoculation  is  not  necessary  for  diagnostic 
purposes. 

Bacillus  of  Bubonic  Plague 

Plague  bacilli  (B.  pestis)  are  found  in  the  sputum  of 
primary  pulmonary  plague,  and  in  the  pneumonia  and 
terminal  pulmonary  oedema  of  severe  plague  septicemia. 
Patients  convalescing  from  pulmonary  plague  may  expec- 
torate virulent  pest  bacilli. 

Microscopical  Examination. — Plague  bacilli  are  found 
in  the  sputum  in  pure  culture,  or  frequently  together  with 


54     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

other  bacteria — namely,  diplococci  and  streptococci.  The 
smears  are  best  fixed,  according  to  Sobernheim,  in  abso- 
lute alcohol,  which  is  dropped  on  the  cover  glass,  allowed 
to  act  about  a  minute,  then  lighted,  and  quickly  extin- 
guished. They  are  stained  with  dilute  borax  methylene 
blue.  The  plague  bacilli  appear  as  short,  oval  rods,  which 
are  stained  more  intensely  at  the  ends  than  in  the  middle 
(polar  staining) .  Their  form,  however,  varies  [greatly. 
In  addition  to  the  typical  rods,  short,  oval  ones  (coccus 
type) ,  as  well  as  long  ones  (rod  type) ,  and  often  involu- 
tion forms  in  the  shape  of  irregularly  bordered  ovoids  or 
discs,  which  stain  poorly,  and  resemble  yeast  cells,  are 
seen.  The  plague  bacillus  is  decolorized  by  Gram. 

Cultivation. — The  reaction  of  the  culture  media  must 
be  neutral  or  slightly  alkaline.  Cultures  on  agar  must  be 
kept  at  30°  C.,  those  on  gelatine  20°  to  22°  C.  The  latter 
is  particularly  suited  to  the  examination  of  sputum  and 
other  secretions  which  contain  other  bacteria  in  addition 
to  the  plague  bacilli,  for  the  plague  bacillus  develops  well 
at  22°  C.,  while  the  growth  of  the  other  bacteria  is  in- 
hibited. Gelatine  plates  are  inoculated  in  the  same  man- 
ner as  are  agar  plates,  by  spreading  the  material  to  be 
examined  in  a  thin  film  over  the  hardened  gelatine.  On 
agar  plates,  after  twenty-four  hours'  growth,  small  colonies 
resembling  dew-drops  are  visible,  which,  after  forty-eight 
hours,  appear  transparent,  with  a  prominent,  darkly  col- 
ored, granular  centre,  and  a  broad,  delicate,  irregular 
periphery.  On  dry  culture  media  containing  8  to  4  per 
cent,  of  sodium  chloride  plague  bacilli  develop  the  charac- 
teristic involution  forms.  On  gelatine,  which  they  do 
not  liquefy,  they  produce,  after  two  to  three  days,  yellow 
colonies,  whose  coarsely  granular  centre  rises  above  the 
surface  of  the  gelatine,  and  is  surrounded  by  a  delicate, 
clear,  jagged  border. 


SPUTUM  55 

The  stalactite  formation  in  undisturbed  bouillon  cul- 
tures is  characteristic. 

Animal  Inoculation. — The  most  suitable  test  animals 
are  rats  and  guinea-pigs.  The  former  are  inoculated  either 
subcutaneously,  or  on  the  uninjured  conjunctiva,  or  by 
means  of  their  food;  the  latter  cutaneously  by  inunction 
on  the  shaved  abdomen.  This  latter  method  gives  espe- 
cially good  results  in  sputum  examination.  After  one  or 
two  days  the  regional  lymph-nodes  become  markedly 
sWollen,  and  after  four  or  five  days  death  ensues.  Material 
for  cultures  can  be  obtained  from  the  buboes  as  early  as 
twenty-four  to  forty-eight  hours  after  the  inoculation. 
The  cultivated  bacteria  are  identified  by  means  of  aggluti- 
nation tests. 

Typhoid  bacilli  are  occasionally  found  in  the  sputum 
.in  bronchitis  and  pneumonia  accompanying  typhoid  fever. 
In  the  cases  in  which  they  are  detected,  they  are  found 
either  alone  or  together  with  streptococci,  diplococci,  and 
influenza  bacilli.  Anthrax  bacilli  appear  in  the  sputum 
of  pulmonary  anthrax  (wool- sorter's  disease).  Bacterium 
coli  accompanying  pneumococci  has  been  very  frequently 
detected  in  cases  of  pneumonia  in  nursing  children. 
These  bacteria  are  identified  according  to  methods  de- 
scribed elsewhere. 

Sputum  which  is  expectorated  in  a  decomposed  condi- 
tion, as  in  pulmonary  gangrene,  bronchiectasis,  and  putrid 
bronchitis,  has  a  rich  bacterial  flora.  Besides  the  true 
exciters  of  inflammation,  B.  fusiformis,  proteus,  pyocya- 
neuSj  pseudo-diphtheria  bacilli,  occasionally  acid-fast 
bacilli,  etc.,  maybe  present. 

Sputum  which  is  expectorated  when  an  empyema  has 
ruptured  into  the  lungs  contains,  usually,  in  addition  to 
other  micro-organisms,  anaerobic  bacteria. 


CHAPTER   V 
EXAMINATION  OF  THE  GASTRIC  CONTENTS 

General  Characteristics 

(a)  Quantity.  —  The  filtrate  of  the  gastric  contents,  ob- 
tained exactly  one  hour  after  the  Ewald  test  breakfast 
(85  to  70  grammes  of  white  bread  and  one  cup  of  tea)  , 
gives  an  idea  of  the  amount  of  the  gastric  contents,  suffi- 
ciently accurate  for  practical  purposes.  This  is  normally, 
according  to  Boas,  20  to  25  cc. 

A  more  accurate  estimation  of  the  total  gastric  contents 
is  carried  out  according  to  Strauss  in  the  following  man^ 
ner  :  First,  a  portion  of  the  gastric  contents  is  withdrawn, 
its  quantity  and  specific  gravity  determined;  a  definite 
quantity  of  water  is  then  introduced  into  the  stomach, 
allowed  to  mix  with  the  gastric  contents,  as  much  as  pos- 
sible withdrawn,  and  the  specific  gravity  of  the  diluted 
gastric  contents  determined.  The  following  formula  gives 
the  desired  quantity: 

.  +  (a-V)S'-a 


v 


S-S' 


in  which  S  represents  the  specific  gravity  of  the  un- 
diluted, S'  the  specific  gravity  of  the  diluted  contents,  V 
the  quantity  of  the  diluted  contents,  and  a,  the  quantity 
of  water  added. 

(b)  The  odor  of  the  gastric  contents  is  normally  slight. 
Even  under  pathological  conditions  there  may  be  no 
marked  odor,  if  the  stomach  was  empty  before  its  recep- 

56 


GASTRIC    CONTENTS  57 


tion  of  the  test  breakfast.  In  cases  of  marked  gastrec- 
tasis,  there  is  often  a  strong  pungent  odor,  due  to  volatile 
fatty  acids  (butyric  acid,  valerianic  acid) .  Decomposi- 
tion of  an  extensive  carcinoma  of  the  stomach  produces  a 
foul  odor;  in  cases  of  ileus  the  odor  is  fecal. 

(c)  Color. — Pure  gastric  juice,  as  well  as  the  gastric 
contents  after  the   test  breakfast,  is  normally  colorless. 
Frequently,  however,  a  slight  mixture  of  bile  causes  a 
yellow  or  greenish  color.     The  presence  of  a  larger  amount 
of  bile  pigment  produces  a  grass-green  color  (the  bilirubin 
being  converted  by  a  longer  stay  in  the  stomach  into  bili- 
verdin) . 

The  change  of  color  in  pathological  cases  is  most  fre- 
quently due  to  the  admixture  of  blood.  Small  streaks  of 
blood  on  the  surface  of  the  gastric  contents  have  no  par- 
ticular meaning,  as  they  are  usually  caused  by  retching. 
A  bloody  coloration  of  the  entire  contents  points  to  the 
presence  of  severe  disease,  and  contraindicates  further 
sounding. 

(d)  Consistency. — The  gastric  contents  after  the  test- 
breakfast  have  usually  a  thin,  pappy  consistency.     When 
mixed  with  large  quantities  of  mucus,  they  have  a  slimy 
consistency. 

Inspection  of  the  gastric  contents  gives  information  as 
to  the  extent  of  the  action  of  the  gastric  juice.  A  dis- 
tinction is  made  between  absolutely  undigested,  partially 
digested,  and  well-digested  gastric  contents.  When  diges- 
tion is  entirely  absent,  the  gastric  contents  resemble  the 
original  meal  lying  in  water.  In  partially  digested  gastric 
contents,  more  or  less  undigested  food  particles  are  visible. 
Occasionally,  the^formation  in  the  receptacle  of  three  strata 
may  be  noticed.  The  top  stratum  consists  usually  of 
mucus  or  coarse  food  particles  (mostly  undigested) ;  the 
middle,  the  largest  stratum,  of  fluid;  the  bottom  of 


58    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

chyme.  In  the  macroscopical  examination  of  the  gastric 
contents  we  must  pay  attention  whether  pus,  blood,  or 
stagnated  food  is  present. 

Qualitative  Chemical  Examination 

1.  Reaction. — The  reaction  of  the  gastric  contents  is 
determined  in  the  usual  manner  with  litmus-paper.  It 
may  be  acid,  neutral,  amphoteric,  or  alkaline. 

In  the  majority  of  normal  and  pathological  cases  the 
reaction  of  the  gastric  contents  is  acid. 

The  normal  acid  reaction  of  the  gastric  contents  after 
the  test-meal  is  due  to : 

1.  Free  hydrochloric  acid. 

2.  Combined  hydrochloric  acid. 

3.  Acid  phosphates. 

4.  Traces  of  organic  acids  (carbonic  acid,  lactic  acid, 
acetic  acid,  butyric  acid,  etc. ) . 

2.  Free  Hydrochloric  Acid. — The  term ( '  free  hydrochloric 
acid"  is  generally  used  in  contradistinction  to  the  term 
1 '  combined  hydrochloric  acid. ' '  The  hydrochloric  acid 
has  a  marked  affinity  toward  albuminoid  substances  and 
their  digestive  products  with  which  it  forms  acid,  loose 
combinations.  In  the  first  stage  of  the  digestion  the 
largest  portion  of  the  secreted  hydrochloric  acid  is  com- 
bined by  albuminoid  substances.  Thus  we  term  free  hy- 
drochloric acid  the  surplus  left  after  the  combination  of 
the  albuminoid  affinities  present. 

The  other  tests  for  free  acids  are  used  also  as  tests  for 
free  hydrochloric  acid,  and  are,  therefore,  described  under 
that  heading. 

The  tests  for  free  hydrochloric  acid  may  be  divided 
into  two  groups : 

(1)  Tests  characteristic  of  hydrochloric  acid  alone. 

(2)  Tests  detecting  all  free  acids,  but  which,  in  the 


GASTRIC    CONTENTS  59 

examination  of  the  gastric  contents,  may  be  used  as  tests 
for  free  hydrochloric  acid. 

To  the  first  group  belongs  Gunsburg's  test  with 
phloroglucin-vanillin. 

The  reagent  consists  of : 

Phloroglucin 2.0 

Vanillin 1.0 

Absolute  alcohol        .       .       .       .30.0 

Three  drops  of  the  reagent  are  thoroughly  mixed  in  a 
porcelain  dish  with  an  equal  quantity  of  filtered  gastric 
contents,  carefully  heated  over  a  small  flame  (without 
reaching  the  boiling-point)  until  the  mixture  has  entirely 
evaporated.  A  beautiful  carmine  mirror  forms,  especially 
at  the  edge.  This  mirror  appears  even  with  a  dilution  of 
0.01  per  cent,  hydrochloric  acid.  With  a  dilution  of 
0.005  per  cent,  merely  fine  red  streaks  are  produced. 
This  reaction  is  not  produced  even  by  the  most  highly 
concentrated  organic  acids.  As  it  is  also  very  delicate,  it 
is  recognized  universally  as  the  surest  and  most  reliable 
reaction  for  the  detection  of  free  hydrochloric  acid. 

According  to  Boas  this  reaction  may  be  carried  out 
with  strips  of  filter-paper  impregnated  with  the  reagent. 
When  such  a  reagent-paper  is  touched  with  2  to  3  drops 
of  gastric  contents  and  carefully  warmed  over  a  flame,  a 
carmine  spot  appears,  which  remains  unchanged  on  the 
addition  of  ether. 

It  must  be  mentioned  that  Gunsburg^s  reagent  decom- 
poses if  kept  a  long  time,  and  it  is,  therefore,  advisable 
to  test  the  solution  with  very  dilute  hydrochloric  acid 
carrying  out  the  reaction. 

Of  the  reactions  of  the  second  group  are  to  be  recom- 
mended : 


60    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

(a)  THE  TEST  WITH  KONGO-PAPER— The  red  color  of 
the  reagent-paper  is  changed  into  blue  by  free  hydrochloric 
acid.     The  more  free  acid  is  present,  the  stronger  becomes 
the  shade  of  blue.     With  but  faint  traces  of  free  hydro- 
chloric acid  the  color  changes  to  light  black-blue. 

(b)  THE  TEST  WITH  METHYL  VIOLET. — Weak  hydro- 
chloric acid  (under  0.5  per  cent.)  colors  a  violet  solution 
of  this  dye  blue.     Organic  acids  change  the  color  of  the 
solution    only    when   more    concentrated    (over   0.5   per 
cent. ) . 

Performance  of  tlie  Test. — To  5  to  10  cc  of  water  in 
a  test-tube  2  to  8  drops  of  a  concentrated  watery  solution 
of  methyl-violet  are  added,  which  must  give  the  water  a 
distinct  violet  color.  To  another  test-tube,  containing 
5  to  10  cc  of  gastric  contents,  the  same  amount  of  dye 
is  added  as  was  added  to  the  water,  and  the  two  solu- 
tions are  compared.  If  the  gastric  contents  appear  blue, 
free  hydrochloric  acid  is  present. 

(c)  THE  TEST  WITH    DIMETHYLAMIDOAZOBENZOL. — A 
0.5  per  cent,  alcoholic  solution  of  this  dye  is  used  as 
reagent.      Hydrochloric    acid   colors   the    orange-yellow 
solution  bright  red. 

Performance  of  the  Test. — To  3  to  5  cc  of  the  filtered 
gastric  contents  3  drops  of  the  solution  are  added.  If  even 
a  faint  trace  of  free  hydrochloric  acid  (0.002  per  cent.) 
is  present,  the  solution  becomes  fiery  red.  Organic  acids 
produce  this  reaction  only  when  more  concentrated  than 
0.5  per  cent.,  and  then  only  in  the  presence  of  albumin, 
peptone,  or  mucus.  Neither  does  loosely  combined  hydro- 
chloric acid  produce  this  reaction. 

These  color  tests  are  more  or  less  sensitive,  but  they 
do  not  give  absolutely  reliable  results,  and  when  hydro- 
chloric acid  is  present  only  in  small  amount,  it  is  hard 
to  distinguish  it  by  them  from  organic  acids. 


GASTRIC    CONTENTS  61 

3.  Lactic  Acid. — Of  the  two  kinds  of  lactic  acid,  fer- 
mentation lactic  acid  (optically  inactive) ,  and  meat  lactic 
acid  (optically  active) ,  the  first  only  need  be  considered  in 
examining  the  gastric  contents.     It  is  formed  as  a  prod- 
uct of  the  fermentation  of  carbohydrates  caused  by  bac- 
teria (Bacterium  acidi  lactici). 

It  is  detected  by  the  following  reactions : 
The  Simple  Ferric  Chloride  Test.— To  20  to  30  cc  of 
water  3  to  5  drops  of  liquor  ferri  sesquichlorati  is  added. 
The  water  assumes  a  hardly  noticeable  yellow  tinge.  The 
thus  obtained  reagent  is  divided  into  two  test-tubes  and 
into  one  of  the  test-tubes  the  gastric  contents,  which  are 
to  be  examined,  are  added,  drop  by  drop.  If  lactic  acid 
is  present,  the  solution  changes  its  color  to  canary- 
yellow.  The  change  of  the  color  becomes  more  visible  by 
holding  the  two  test-tubes  against  a  white  background. 

(b)  Modification  According  to  H.  Strauss. — Five  cc 
of  gastric  contents  are  shaken  with  20  cc  of  alcohol-free 
ether.  After  the  solution  has  settled,  1  part  (5  cc)  of 
the  ether  is  poured  off,  diluted  with  4  parts  of  water  and 
2  drops  of  a  ferric  chloride  solution  (1  :  9),  and  vigor- 
ously shaken.  When  about  0.1  per  cent,  of  lactic  acid 
is  present  a  distinct  green  color  appears,  when  less  is 
present  a  fainter  green  color  appears. 

4.  Volatile  Fatty  Acids. — Of  the  volatile  fatty  acids, 
particularly  acetic  and  butyric  acids  are  to  be  considered 
in  the  examination  of  the  gastric  contents.     They  are 
either  introduced  with  the  food  or  are  formed  as  products 
of  abnormal   carbohydrate   fermentation.      It  is  only  in 
the  latter  case  that  they  are  of  diagnostic  significance. 

As  a  preliminary  test  for  the  presence  of  volatile  fatty 
acids  the  following  simple  test  (adequate  for  practical 
purposes)  may  be  used.  About  10  cc  of  the  gastric 
contents  are  heated  in  a  test-tube,  at  the  upper  end  of 


62    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

which  there  is  a  small  strip  of  moist  blue  litmus-paper. 
When  volatile  acids  are  present  the  litmus-paper  turns  red 
(Leo). 

The  following  method  is  more  definite:  To  15  to  20 
cc  of  gastric  contents  1  gramme  of  sodium  sulphate  is 
added,  and  the  mixture  thoroughly  shaken  two  or  three 
times,  each  time  with  50  cc  of  ether.  The  ether  is 
poured  off  and  distilled.  A  fluid  residue  remains,  which, 
if  organic  acids  are  present,  has  a  distinct  acid  reaction 
and  a  characteristic  odor.  The  residue  is  then  divided 
into  two  equal  portions,  with  which  the  tests  for  acetic 
and  butyric  acids  are  carried  out. 

(a)  Detection  of  Acetic  Acid. — The  liquid  is  taken  up 
with  water,  neutralized  exactly  with  a  dilute  soda  solu- 
tion, and  a  drop  of  ferric  chloric  added.  If  acetic  acid  is 
present  the  liquid  turns  blood-red,  and,  when  boiled, 
throws  down  a  brownish-red  precipitate  of  basic  ferric 
acetate. 

To  be  sure,  formic  acid  produces  the  same  reaction, 
but  this  fact  does  not  alter  the  diagnostic  value  of  a  posi- 
tive reaction,  since  formic  acid  can  be  present  in  the 
gastric  contents  only  as  a  product  of  acid  fermentation. 

(#)  Detection  of  Butyric  Acid. — The  second  portion  of 
the  ether  residue  is  dissolved  in  2  to  3  drops  of  water, 
and  treated  with  a  very  small  particle  of  calcium  chloride. 
The  butyric  acid  separates  (because  of  its  insolubility  in 
salt  solutions)  into  little  drops  which  float  on  the  surface, 
and  which  have  the  characteristic  odor  of  butyric  acid. 

5.  Pepsin  and  Pepsinogen. — Pepsin,  the  proteolytic  fer- 
ment of  the  gastric  juice,  is  formed  from  pepsinogen,  the 
specific  product  of  the  peptic  cells  of  the  gastric  glands, 
by  the  action  of  acids.  The  conversion  of  pepsinogen 
into  active  pepsin  is  produced  with  special  rapidity  by 
the  action  of  hydrochloric  acid. 


GASTRIC    CONTENTS  63 

Upon  this  fact  depends  the  detection  of  pepsin  and 
pepsinogen.  If  the  gastric  contents  contain  free  acid, 
and  at  the  same  time  digest  albumin,  pepsin  is  present. 
When  the  gastric  juice  contains  no  free  acid,  only  pep- 
sinogen can  be  present.  Such  gastric  contents  must  have 
the  power  to  digest  albumin  after  the  addition  of  a  suffi- 
cient quantity  of  hydrochloric  acid.  If  this  is  not  the 
case,  pepsinogen  is  also  absent. 

Performance  of  the  Digestive  Test. 

1.  According  to  Mett.    Egg-albumin  is  filtered  through 
a  piece  of  gauze  into  a  small  beaker  or  wide  test-tube,  and 
short  glass  tubes  having  a  lumen  of  about  two  millimetres 
are  slowly  dropped  into  it.     Air-bubbles,  which  rise  in  the 
tubes,  are  allowed  to  escape,  aided  by  gentle  tapping.     The 
vessel  containing  the  tubes  is  then  placed  in  a  boiling- 
water  bath  for  five  to  ten  minutes.      The  flame  is  then 
removed,  and  the  glass  allowed  to  cool  slowly  for  several 
hours.     The  test-tube  is  now  broken,  and  the  small  tubes 
which  are  filled  with,  and  imbedded  in,  the  coagulated 
egg-albumin,  are  cut  out  and  preserved  either  in  glycerine 
or  chloroform  water. 

One  tube  is  used  for  each  test.  It  is  first  washed 
with  water,  then  put  into  a  test-tube  containing  10  cc 
of  filtered  gastric  contents,  and  placed  in  an  incubator  for 
twenty- four  hours.  If  during  the  chemical  examination 
hydrochloric  acid  was  found  to  be  absent,  1  or  2  drops  of 
official  hydrochloric  acid  are  added;  if  pepsin  is  present, 
a  portion  of  the  albumin  will  be  digested  at  the  end  of 
twenty-four  hours.  This  test  serves  at  the  same  time 
for  the  quantitative  estimation  of  pepsin,  the  length  of 
the  digestive  column  of  egg-albumin  (in  millimetres) 
being  proportional  to  the  amount  of  pepsin. 

2.  JacoWs  method.    The  principle  of  the  test  consists 
in  the  fact  that  a  ricin  solution,  which  is  made  cloudy  by 


64     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

the  addition  of  HC1,  is  clarified  by  the  addition  of  pep- 
sin. First  several  solutions  are  prepared  of  filtered  gas- 
tric juice,  which  have  been  previously  diluted  with  water 
and  the  lowest  degree  of  dilution  is  determined  at  which 
a  certain  quantity  of  ricin  solution  will  clarify. 
Reagents  needed: 

1.  A  1  per  cent,   ricin  solution    in    a   5    per   cent. 
NaCl  solution  (0.5  ricin  in  50  cc  salt  solution  prepared 
extemporaneously) . 

2.  A  decinormal  HC1  solution. 

Procedure. — Eight  test-tubes  of  equal  calibre  are  filled 
each  with  2  cc  of  the  slightly  cloudy  and  filtered  ricin 
solution  and  with  0.5  cc  of  a  decinormal  HC1  solution. 
The  addition  of  the  HC1  produces  a  milky  cloudiness. 
The  test-tubes,  numbered  1  to  8,  are  put  in  a  test-tube- 
holder.  Tubes  1  and  8  are  for  controlling  purposes, 
2  to  7  contain  the  gastric  juice  in  the  various  degrees  of 
dilution  as  follows : 

Tube  2.   1.0  cc  of  the  undiluted  gastric  juice  (dilution 

1:1) 
Tube  8.  0.2  cc  of  the  undiluted  gastric  juice   (dilution 

(1:1) 
Tube  4.   1.0  cc   10  times  diluted  gastric  juice  (dilution 

1:10) 
Tube  5.  0.2  cc  10  times  diluted  gastric  juice  (dilution 

1:50) 
Tube  6.   1.0  cc  100  times  diluted  gastric  juice  (dilution 

1:100) 
Tube  7.  0.5  cc  100  times  diluted  gastric  juice  (dilution 

1:200) 

In  order  to  obtain  equal  volumes,  to  each  test-tube 
distilled  water  is  added  whatever  is  missing  to  3.0  cc :  In 
tube  1,  1.0  cc;  3,  0.8  cc;  5,  0.8  cc;  7,  0.5  cc;  8,  1.0  cc. 


GASTRIC    CONTENTS  65 

A  few  drops  of  pepsin  are  added  to  test-tube  1,  afterward 
^11  test-tubes  are  corked,  shaken  up  and  put  in  the  in- 
cubator for  three  hours  (37°  C. ) ,  after  which  the  test-tubes 
are  read  off.  The  contents  in  tube  1  must  remain  clear, 
in  tube  8  they  must  be  cloudy.  Then  it  must  be  ascer- 
tained at  what  dilution  a  noticeable  clarification  is  effected. 
In  order  to  obtain  standard  solutions  for  comparison  Solms 
arbitrarily  puts  down  that  1  cc  of  gastric  juice  contains 
100  pepsin-units,  which  is  just  about  sufficient  to  clarify 
the  richin  solution  after  three  hours  in  the  incubator  for 
1  cc  of  a  100  times  diluted  gastric  juice.  Normal  gastric 
juice  contains  100  to  200  pepsin  units.  In  case  of  hyper- 
acidity dilutions  up  to  1: 10,000  must  be  employed. 

6.  Renin  and  Reninogen. — Reninogen  is,  like  pepsinogen, 
a  product  of  the  gastric  glands,  and  is  converted  by  the 
action  of  hydrochloric  acid  into  renin.  Renin  possesses 
the  property  of  coagulating  milk  without  the  aid  of  the 
acids  of  the  stomach,  and,  in  fact,  in  the  presence  of  a 
slightly  acid  or  neutral  reaction.  The  action  of  the  fer- 
ment is  absent  when  the  reaction  is  slightly  alkaline,  but 
appears  upon  the  addition  of  solutions  of  calcium  salts. 
The  action  of  the  calcium  salts  is  explained  by  the  fact 
that  they  convert  reninogen  into  renin. 

Detection  of  Renin. — Ten  cc  of  the  filtered  gastric  con- 
tents are  exactly  neutralized  with  weak  sodium  hydrate 
solution  (0.5  per  cent. ),  and  mixed  with  an  equal  quan- 
tity of  neutral  or  amphoteric  boiled  milk.  The  mixture  is 
placed  in  an  incubator.  When  renin  is  present  the  casein 
will  coagulate  within  ten  to  thirty  minutes,  and  after 
longer  staying  will  form  a  single  coagulum  (cheese) .  It 
must  be  determined  each  time  after  the  coagulation  that 
the  reaction  of  the  mixture  is  unchanged,  since  the  casein 
may  have  been  coagulated  by  acids  which  have  developed 
late. 


66     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Detection  of  Reninogen. — To  2  cc  of  the  filtered  gastric 
contents  are  added  an  excess  of  sodium  carbonate,  2  cc  of 
a  3  per  cent,  calcium  chloride  solution,  and  10  cc  of  milk, 
and  the  mixture  placed  in  an  incubator.  If  reninogen  is 
present  coagulation  will  gradually  take  place. 

According  to  Boas  the  quantitative  analysis  for  rennet 
depends  upon  the  principle  of  how  far  the  gastric  juice 
can  be  diluted  without  losing  its  coagulating  power  of 
milk.  The  filtered  gastric  contents  are  first  neutralized 
with  sodium  hydrate.  One  cc  is  taken  up  with  the  pipette 
and  is  diluted  with  ten  times  its  volume  of  water,  half  of 
this  is  again  diluted  with  an  equal  volume  of  water.  We 
proceed  in  the  same  way  and  prepare  (50  cc  every  time) 
dilutions  of  1  in  10,  1  in  20,  1  in  40,  1  in  80,  1  in  320, 
etc.  Into  each  test-tube  that  contains  5  cc  of  the  thus 
diluted  gastric  juice  is  added  5  cc  lukewarm,  boiled  milk 
and  2J  cc  of  a  1  per  cent,  solution  of  chlorcalcium.  The 
test-tubes  are  shaken  up  a  few  times  and  put  into  the  in- 
cubator or  into  a  warm-water  bath  of  40°  C.  After  fifteen 
to  twenty  minutes  we  observe  at  which  lowest  dilution 
coagulability  is  still  seen.  In  normal  secretion  a  dilu- 
tion of  1:160  shows  firm  coagula,  while  in  the  next  dilu- 
tion of  1  in  320  only  a  fine  flocculent  precipitate  is  seen. 
In  cases  of  hypersecretion  we  get  positive  results  even 
in  a  dilution  of  1  in  800. 

The  fermentation- tests  for  rennet  and  pepsin  are  to  be 
made  for  diagnostical  purposes  only,  in  such  gastric  con- 
tents which  contain  no  free  hydrochloric  acid. 

Bile  Pigment — The  tests  for  bile-pigment  are  made  ac- 
cording to  the  methods  of  Gmelin  or  Rosin.  (Cf .  Chapter 
VII.) 

8.  Blood — The  tests  for  blood  are  made  by  way  of 
chemistry,  the  microscope  and  by  the  spectroscope.  The 
simplest  chemical  reactions  are : 


GASTRIC    CONTENTS  67 

(a)  Guaiacum  Test  (According  to  Weber). — A  f ew  cc 
of  glacial  acetic  acid   are  added  to  10  cc  of   the  gastric 
contents  and  shaken  with  the  same  amount  of  ether.    After 
a  sediment  has  formed  a  few  cc  of  the  etheric  extract  are 
poured  off  and  mixed  with  20  drops  of  resinous  turpentine 
or   hydrogen-superoxide.     Then   a   fresh-made   alcoholic 
guaiacum  solution  is  added,  drop  by  drop,  while  shaking. 
The  solution  must  not  be  too  concentrated;   too  much 
guaiacum  mars  the  test,  one  must  be  guided  by  the  color, 
which  is  to  be  brownish-yellow,  but  not  dark-brown.     The 
color  changes  to  blue-violet  if  blood  is  present,  but  in  the 
absence  of   blood-pigment  to  red-brown;    after  dilution 
with  water  the  blue  pigment  can  be  extracted  with  chloro- 
form. 

(b)  The  Aloin   Test. — The   tincture  of   guaiacum    is 
substituted  by  a  fresh-made  aloin  solution  (the  tip  of  a 
knife  of  aloin  in  10  cc  alcohol) .     The  color  changes  in- 
stantly or  after  some  time  to  strawberry-red. 

(c)  The  Benzidin  Test  (is  to  be  used  only  when  there 
are  no  rests  of  meat  in  the  gastric  contents) .     In  a  test- 
tube  is  put  benzidin — a  tip  of  a  knife,  1  cc  glacial  acetic 
acid,  2  cc   hydrogen-superoxide.     In  the  other  test-tube 
a  few  cc  of  the  gastric  contents  are  heated  to  the  boiling- 
point,  3  to  5  drops  from  the  second  tube  are  poured  into 
the  first  tube  and  shaken.     If  blood-pigment  is  present, 
the  color  becomes  emerald-green-  or  green-blue. 

This  test  is  extremely  sensitive. 

The  spectroscopical  test  is  made  in  the  same  way  as 
the  examination  of  the  urine  (cf.  ibidem). 

When  the  gastric  contents  contain  free  hydrochloric 
acid  and  a  large  quantity  of  organic  acids,  oxyhsemoglobin 
is  converted  into  hsematin  chloride.  The  latter  is  only 
slightly  soluble  in  water.  Therefore  under  these  condi- 
tions the  spectroscopical  examination  may  yield  a  negative 


68     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

result  even  when  a  large  quantity  of  blood  is  present.  It 
is  well  in  such  cases  to  treat  the  gastric  contents,  accord- 
ing to  Weber,  with  a  few  cc  of  concentrated  acetic  acid, 
and  shake  thoroughly  with  ether.  When  blood  is  present 
the  gastric  contents  assume  a  reddish-brown  color,  and 
show  the  spectrum  of  haematin  chloride. 

9.  Hydrogen  sulphide  is  easily  recognized  by  its  char- 
acteristic odor.  It  can,  in  addition,  be  detected  by  the 
following  simple  test: 

A  strip  of  paper,  saturated  with  an  alkaline  solution 
of  lead  acetate,  is  fastened  in  a  cork.  The  vessel  con- 
taining the  gastric  contents  is  tightly  closed  with  this 
cork  so  that  the  strip  of  paper  is  entirely  within  the 
vessel.  When  hydrogen  sulphide  is  present  the  paper 
turns  black. 

Quantitative  Chemical   Examination  of  the  Gastric 
Contents 

1.  Estimation  of  Total  Acidity. — In  the  estimation  of 
the  total  acidity  all  acid-reacting  substances  in  the  gastric 
contents  come  into  consideration: 

(a)  Free  and  combined  hydrochloric  acid. 

(b)  Free  and  combined  organic  acids  (lactic,  butyric, 
and  acetic  acid). 

(c)  Acid  phosphates. 

The  acidity  is  expressed  by  the  quantity  of  decinormal 
alkali  solution  which  must  be  added  to  100  cc  of  the  gas- 
tric contents  in  order  to  neutralize  them. 

Procedure. — To  10  cc  of  the  filtered  gastric  contents 
in  a  small  beaker,  1  or  2  drops  of  an  alcoholic  solution  of 
phenolphthalein  are  added.  Decinormal  alkali  solution 
is  run  into  it  from  a  Mohr's  burette,  with  vigorous  shak- 
ing, until  the  liquid  assumes  a  distinct  red  color.  The 


GASTRIC    CONTENTS  69 

level  of  the  liquid  in  the  burette  is  noted  before  and  after 
the  titration.  The  amount  of  decinormal  alkali  solu- 
tion used  is  determined  by  subtraction  and  multiplied 
by  10. 

2.  Estimation  of  Free  Hydrochloric  Acid. 

(a)  ACCORDING  TO  MINZ. — According  to  this  method, 
the  gastric  contents  are  treated  with  decinormal  alkali 
solution  until  the  reaction  for  free  hydrochloric  acid  just 
disappears. 

Procedure. — Ten  cc  of  the  filtered  gastric  contents  are 
titrated  in  a  beaker  with  decinormal  alkali  solution.  At 
first  the  solution  is  added  1  cc  at  a  time,  and  after  the  ad- 
dition of  each  cc,  Giinsburg's  reaction  is  carried  out  with 
a  drop  of  the  solution.  The  titration  is  proceeded  with  in 
this  manner  until  Gunsburg's  reaction  disappears.  The  ap- 
proximate amount  of  decinormal  alkali  solution  needed  to 
neutralize  the  free  hydrochloric  acid,  which  is  so  ob- 
tained, is  then  rendered  more  accurate  by  adding  to  an- 
other 10  cc  of  the  gastric  contents,  1  cc  less  of  the  deci- 
normal alkali  solution  than  was  previously  used,  and 
proceeding  with  the  titration,  adding  the  alkali  1  drop  at 
a  time.  After  every  second  drop,  Gunsburg^s  reaction  is 
carried  out. 

If  it  is  found,  for  example,  that  after  the  addition  of 
2.5  cc  the  reaction  is  still  present,  while  after  the  addition 
of  2.6  cc  it  is  absent,  the  amount  of  free  acid  is  2.6  X  10 
=  26  cc  decinormal  alkali  solution  (calculated  for  100  cc 
of  gastric  contents).  Each  cc  of  the  decinormal  alkali 
solution  represents  0.00365  gramme  of  hydrochloric  acid. 
The  percentage  in  this  case  is,  therefore,  0.00365  X  26  = 
0.0949  per  cent.  This  method  gives  reliable  and,  for 
practical  purposes,  thoroughly  useful  results. 

(b)  ACCORDING     TO     TOEPFER. — According    to    this 
method  the  free  hydrochloric  acid  is  estimated,  using  a 


70    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

0.5  per  cent,  alcoholic  solution  of  dimethylamidoazobenzol 
as  an  indicator. 

Procedure. — To  10  cc  of  the  filtered  gastric  contents, 
2  to  3  drops  of  the  dimethylamidoazobenzol  solution  are 
added;  to  the  now  bright  red  liquid  decinormal  alkali 
solution  is  added  from  a  burette  until  the  red  color  of  the 
fluid  entirely  disappears,  giving  place  to  the  original  yel- 
low color.  This  method  gives  comparatively  reliable  re- 
sults only  when  large  amounts  of  hydrochloric  acid  and 
very  small  amounts  of  organic  acids  are  present.  Under 
the  opposite  conditions,  it  gives  very  inaccurate  results, 
as  the  organic  acids  are  included  in  the  titration. 

3.  Estimation  of  Total  Hydrochloric  Acid  According  to 
Toepfer. — The  quantity  of  total  hydrochloric  acid  is  com- 
puted from  its  components,  the  free  and  combined  hydro- 
chloric acid. 

The  free  hydrochloric  acid  is  estimated,  according  to 
the  above-described  method,  by  titration  with  dimethyl- 
amidoazobenzol, combined  in  the  following  manner: 

Ten  cc  of  the  filtered  gastric  contents  are  titrated  with 
decinormal  alkali  solution,  3  to  4  drops  of  a  1  per  cent, 
watery  solution  of  alizarin  sulphonate  of  sodium  being 
added  as  an  indicator,  until  the  originally  yellow  liquid 
passes  through  red  into  a  pure  violet.  Since  alizarin  reacts 
with  all  the  factors  of  acidity  except  combined  hydrochloric 
acid,  the  subtraction  of  the  acidity  found  in  this  manner 
from  the  total  acidity  gives  the  amount  of  combined  hy- 
drochloric acid.  Example:  In  the  titration  of  10  cc  of 
filtered  gastric  contents  with  alizarin  sulphonate  of  so- 
dium, 4.5  cc  of  decinormal  alkali  solution  are  used — i.e., 
45.0  cc  to  100  cc.  The  total  acidity  was  previously  esti- 
mated, and  amounted  to  50.0  cc  of  the  decinormal  alkali 
solution.  The  acidity  due  to  combined  hydrochloric  acid 
is,  therefore,  50  —45  =  5.0.  If  this  number  is  multiplied 


GASTRIC    CONTENTS  71 

by  0.00865,  the  percentage  of  combined  hydrochloric  acid 
is  obtained:  5  X  0.00365  =  0.018  per  cent.  Granted  that 
the  quantity  of  free  hydrochloric  acid  (estimated  by  titra- 
tion  with  dimethylamidoazobenzol  as  indicator)  =0.15  per 
cent.,  then  the  total  hydrochloric  acid  amounts  to  0.15  + 
0.018  =  0. 168  per  cent.  By  subtracting  the  acidity  due  to 
free  and  combined  hydrochloric  acid  from  the  total  acid- 
ity, the  acidity  due  to  organic  acids  and  acid  phosphates 
can  be  determined. 

4.  Estimation  of  Lactic  Acid— According  to  Leo,  10  cc 
of  the  filtered  gastric  contents  are  boiled  until  the  escaping 
steam  no  longer  reddens  a  moistened  strip  of  blue  litmus- 
paper.  The  liquid,  which  has  in  this  manner  been  freed 
from  volatile  acids,  is  then  extracted  with  ether  six  times, 
50  cc  of  ether  being  used  each  time.  The  ethereal  extracts 
are  poured  together,  and  the  ether  distilled  or  driven  off  on  a 
water-bath.  The  residue  is  taken  up 'in  a  small  quantity 
of  water  and  titrated  with  decinormal  alkali  solution,  2  to 
8  drops  of  phenophthalein  being  added  as  an  indicator. 
Each  cc  of  decinormal  alkali  solution  used  represents 
0.009  gramme  of  lactic  acid. 

According  to  Meliring  and  Calm  lactic  acid  and  the 
volatile  fatty  acids  can  be  determined  with  the  same  portion 
of  gastric  contents.  A  measured  quantity  of  the  filtered 
gastric  contents  is  distilled.  The  volatile  acids  which  have 
gone  over  in  the'distillate  are  estimated  by  titration,  while 
the  residue  is  shaken  repeatedly  with  ether.  The  lactic 
acid  in  the  combined  ethereal  extracts  is  estimated  in  the 
same  manner  as  in  Leo's  method. 

Microscopical  Examination  of  the  Gastric  Contents 

For  microscopical  examination,  the  gastric  contents 
are  allowed  to  settle,  a  email  portion  of  the  precipitate 


72     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

withdrawn  with  a  pipette,  and  specimens  made  in   the 
usual  manner. 

Under  normal  conditions  the  microscopical  examina- 
tion of  the  gastric  contents  after  EwalcTs  test-meal  shows 
numerous  starch-granules,  isolated  yeast-cells,  epithelium 
from  the  oral  cavity,  a  little  mucus  or  particles  of  swal- 
lowed sputum.  For  diagnosis  these  constituents  of  the 
chymus  are  of  no  value,  only  the  microscopical  examina- 
tion of  the  contents  of  the  empty  stomach  furnishes  ma- 
terial for  diagnostical  purposes.  If  HC1  is  present  in  the 
empty  stomach,  the  following  constituents  are  found  during 
the  microscopical  examination : 

1.  Nuclei  of  leucocytes  and  epithelium  (the  protoplasm 
is  digested). 

2.  Mucus  of  distinctly  striate  structure. 

3.  Spiral-cells,  i.e.,  snakelike  formations,  originating 
from  the  myelin  of  the  swallowed  sputum  under  the  influ- 
ence of  the  HC1. 

These  three  constituents  are  found  with  normal  secre- 
tion and  with  hypersecretion,  but  no  food-rests  in  the 
stomach  contents.  If,  besides  these  constituents,  food-rests 
are  found  in  the  empty  stomach,  a  stagnation  is  present. 
Besides  the  food-rests  such  as  starch-granules,  muscle 
fibres,  drops  of  fat,  crystals  of  fatty  acids,  rests  of  vege- 
tables, etc.,  there  are  found  numerous  sarcinse  or  yeast- 
cells.  The  sarcina  appears  in  the  stomach  contents  in  two 
different  forms:  1.  In  form  of  bales.  2.  In  irregular 
lumps  or  in  form  of  cubes.  Characteristic  for  the  sarcina 
is  the  cellulose  reaction;  it  changes  to  red- violet  after 
a  chlor-zinc-iodine  solution  is  added  (chlor-zinc,  20.0; 
potassium  iodine,  6.5;  iodine,  1.3;  water,  10.5).  The 
yeast-fungi  appear  as  oval,  quite  highly  refracting,  often 
pearl-necklacelike  arranged  cells,  which  are  readily  dis- 
tinguished from  the  small  starch-granules  by  adding  a 


GASTRIC    CONTENTS  73 

iodine-potassium-iodine  solution  (LugoPs);  starch  colors 
blue,  yeast-fungi  color  yellow.  In  the  absence  of  HC1 
and  other  free  acids  (achylia  gastrica,  gastritis  simplex) 
mostly  unchanged  epithelia  are  found  in  the  empty  stom- 
ach and  isolated  leucocytes,  sometimes  amoebae  and  infu- 
soria. In  malignant  diseases  of  the  stomach  (tumors) 
also  red  blood-corpuscles  and  many  pus-corpuscles  are 
found. 

Schizomycetes  are  only  found  when  they  are  present  in 
very  large  numbers  and  entirely  obscure  the  microscopical 
field.  Of  the  kinds  of  bacteria,  Boasts  ' '  faden  (thread) 
bacilli"  are  quite  frequently  found  in  carcinoma  of  the 
stomach.  They  appear  as  long,  slightly  motile  rods,  lying 
usually  at  an  angle  to  one  another.  They  are,  to  be  sure, 
not  pathognomonic  of  carcinoma  of  the  stomach,  but  they 
are  found  in  nearly  75  per  cent,  of  the  cases.  They  are 
also  present  in  cases  in  which  there  is  stagnation  of  the 
gastric  contents  with  the  absence  of  free  hydrochloric  acid 
and  production  of  lactic  acid. 

Crystalline  bodies  are  comparatively  rare  in  the  gastric 
contents.  The  following  have  been  described:  Leucin 
and  tyrocin  crystals  (in  stagnation),  triple  phosphate 
crystals  and  crystals  of  magnesium  phosphate  (only  in 
alkaline  or  neutral  gastric  secretions),  and,  very  rarely, 
cholesterin  crystals.  For  the  identification  of  the  crystals 
micro-chemical  reactions  are  best  used  (cf.  Microscopy  of 
Urine). 


CHAPTER   VI 
EXAMINATION  OF  THE  FAECES 

General  Characteristics 

1.  Color. — Under  normal  conditions,  in  the  adult, 
hydrobilirubin  is  the  characteristic  fecal  pigment:  bili- 
rubin  is  present  normally  only  in  the  faeces  of  nursing 
children.  The  coloring  of  the  faeces  is  not  due  to  pig- 
ments alone,  but  is  influenced  by  a  great  number  of  fac- 
tors, of  which,  as  a  rule,  the  character  of  the  food  is  the 
most  important.  Under  a  mixed  diet  the  faeces  are  yel- 
lowish-brown in  color,  under  a  meat  diet,  dark  to  blackish- 
brown,  and  under  an  exclusive  milk  diet,  orange  to  light 
yellow.  Foods  which  have  a  peculiar  color  of  their  own 
may  produce  a  characteristic  coloration  of  the  faeces.  For 
example,  following  the  liberal  ingestion  of  chlorophyllic 
vegetables  or  of  lettuce,  the  faeces  may  be  stained  green; 
following  that  of  ' '  blutwurst, ' '  blackish-brown ;  and  that 
of  cocoa,  blackish-red.  Black  cherries  and  blackberries 
stain  the  faeces  blackish-red;  red  wine  and  blueberries, 
reddish-brown  with  a  tinge  of  green.  Drugs  also  fre- 
quently cause  a  characteristic  coloration  of  the  faeces. 
The  green  coloration  following  the  use  of  calomel  is  very 
well  known.  It  is  due  to  the  conversion  of  bilirubin, 
within  the  intestinal  tract,  into  biliverdin.  Following 
the  use  of  bismuth  the  faeces  are  colored  black.  The  col- 
oration is  due  to  the  reduction  of  bismuth  subnitrate 
to  black  oxide  of  bismuth.  Preparations  of  iron  also  fre- 

74 


FAECES  75 

quently  produce  a  black  coloration  of  the  faeces,  which  is, 
however,  limited  to  the  surface. 

In  pathological  conditions  the  pathological  products 
of  the  intestinal  wall  may  influence  the  color  of  the  faeces 
more  or  less.  Thus,  the  liberal  admixture  of  mucus  or 
pus  may  produce  a  grayish- white  to  yellowish-gray  color. 
Blood,  depending  upon  the  quantity  and  upon  the  degree 
of  alteration  of  the  haemoglobin,  may  produce  a  bright-red 
to  pitch-black  coloration.  Bacteria  may  also  produce  a 
characteristic  coloration  of  the  faeces.  It  has  been  possible 
to  cultivate  from  the  stools  of  nursing  infants  and  children 
a  bacillus  ("bacille  de  la  diarrhee  verte  des  enfants," 
Lesage)  whose  cultures  contain  a  pigment  which  colors 
the  faeces  green.  Bacillus  pyocyaneus  may  also,  under 
certain  conditions,  produce  a  green  coloration  of  the  faeces. 

As  the  result  of  obstruction  of  the  bile-duct  (by  catar- 
rhal  swelling,  gall-stones,  tumors,  ascaris,  etc.),  the  stools 
are  clay-colored  (acholous),  and  contain  considerable  fat. 
In  cases  of  intestinal  hyperperistalsis,  with  excessive  diar- 
rhoea, unaltered  bile-pigment  (biliverdin)  may  stain  the 
fasces  green. 

2.  Consistency  and  Form. — According  to  the  consistency 
a  distinction  is  made  between  firm  or  formed,  thick  or 
thin-pasty,  and  watery  stools.     Following  a  chiefly  animal 
diet  the  stools  are,  as  a  rule,  cylindrical  and  firm.     Fol- 
lowing  a  vegetable   diet  they   are   usually   thick-pasty. 
The  firm  faeces  have  occasionally  a  "pencil  form"  (in 
stenosis  or  spasm  of  the  large  intestine),  or  the  so-called 
"sheep  manure"  form.     In  the  latter  case,  round  balls 
the  size  of  a  hazel-nut  are  evacuated.     Thin-pasty  and 
watery  stools  are  usually  pathological.   Following  marked 
hemorrhage  in  the  upper  bowel  or  stomach  the  faeces  have 
a  black,  tarry  appearance. 

3.  Odor. — The  odor  of  the  faeces  is,  under  normal  con- 


76     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

ditions,  due  principally  to  the  presence  of  skatol,  a  prod- 
uct of  the  decomposition  of  albuminoid  substances. 
Indol,  which  is  produced  at  the  same  time,  has  a  lesser 
influence  upon  the  odor  of  the  stools. 

The  odor  of  the  fseces,  therefore,  depends  upon  the  diet 
and  the  degree  of  decomposition  in  the  intestinal  tract. 
Following  a  meat  diet,  rich  in  albumin,  the  fecal  odor  is 
much  more  marked  than  following  a  vegetable  diet,  and 
in  atonic  conditions  of  the  intestines  it  is  stronger  than 
when  intestinal  peristalsis  is  normal.  Under  an  exclusive 
milk  diet  the  odor  is  very  slight,  and  therefore  the  normal 
stool  of  the  nursing  child  is  practically  odorless.  Every 
foul-smelling  stool  from  a  nursing  child  must,  therefore, 
be  considered  pathological. 

In  acute  and  chronic  diarrhoea  the  stools  are  often 
odorless.  The  characteristic  rice-water  stools  of  Asiatic 
cholera  are  also,  as  a  rule,  odorless.  The  evacuations  in 
amoebic  dysentery  have  a  characteristic  gluey  odor. 
Acholous  stools  are  of  themselves  nearly  odorless.  They 
possess  a  foul  odor  only  when  decomposition  resulting 
from  atony  of  the  intestines  accompanies  the  absence  of 
bile.  The  stools  are  fetid  and  foul  smelling  in  cases  of 
ulcerating  and  decomposing  carcinoma  of  the  rectum. 

4.  Macroscopical  Constituents. — A  superficial  examina- 
tion does  not  suffice,  as  a  rule,  for  the  detection  of  the 
macroscopical  constituents  of  the  fseces.  For  this  purpose 
watery  stools  must  be  poured  into  shallow  dishes,  while 
thick-pasty  and  firm  stools  must  first  be  carefully  stirred 
with  a  glass  rod  in  a  large  quantity  of  water.  The 
macroscopical  constituents  are  best  collected  by  means  of 
the  fecal  sieve  suggested  by  Boas.  This  consists  (Fig. 
11)  of  two  hemispheres,  which  are  held  together  by 
means  of  a  bayonet  catch,  and  can  easily  be  taken  apart. 
The  lower  hemisphere  contains  an  exceptionally  fine  sieve 


FAECES  77 

(S),  upon  which  the  faeces  are  spread  out.  The  upper 
hemisphere  has  a  nozzle  for  a  tube,  by  which  it  may  be 
connected  with  any  water-spout,  and  a  chain  with  which 
to  hang  it  from  the  spout.  The  water  is  carefully  turned 
on,  and  a  continuous  fine  stream  allowed  to  flow  over  the 
faeces. 

In  the  upper  hemisphere  is  an  opening  (0),  with  a 
removable  cap,  through  which  a  glass  rod  may  be  intro- 


FIG.  11 

duced,  with  which,  during  the  washing,  the  faeces  are 
stirred  to  a  pasty  mass.  •  The  water  escapes  through  a  pipe 
in  the  lower  hemisphere.  This  procedure  takes  fifteen  to 
thirty  minutes,  and  only  the  coarser  constituents  of  the 
faeces  remain  upon  the  sieve. 

The  macroscopical  constituents  of  the  faeces  especially 
to  be  noticed  are : 

1.  Undigested  food  particles. 

2.  Pathological  products  of  the  intestinal  wall. 
8.  Intestinal  parasites. 


78     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

4.  Gall-stones  and  enteroliths. 

5.  Objects  which  have  been  accidentally  swallowed. 
Of  the  constituents  of  animal  diet,  normally  only  those 

which  are  indigestible,  as  cartilage  and  tendon,  and  pieces 
of  bone  which  have  been  accidentally  swallowed,  are  found 
in  the  faeces.  The  presence  of  pieces  of  muscle  or  connec- 
tive tissue  when  meat  has  been  properly  prepared  and 
not  ingested  in  very  great  quantity  is  considered  patho- 
logical. 

Of  the  vegetable  foods,  white  bread,  potatoes,  fari- 
•naceous  foods,  and  juicy  fruits  (without  the  peels),  leave 
no  undigested  portions  which  can  be  recognized  macro- 
scopically.  Raw  vegetables  (cucumbers,  lettuce,  onions, 
radishes,  asparagus,  and  string  beans)  and  numerous 
fruits  (cranberries,  nuts,  and  currants)  pass  through  the 
intestinal  canal  and  appear  in  the  faeces  practically  un- 
changed. Cooked  fruits  and  vegetables  are  very  much 
more  easily  digested;  and,  as  a  rule,  only  the  poorly 
cooked  and  insufficiently  masticated  portion  can  be  recog- 
nized in  the  faeces  macroscopically.  At  any  rate,  no  diag- 
nostic conclusions  can  be  drawn  from  the  appearance  in 
the  faeces  of  undigested  particles  of  vegetable  matter. 

Of  the  products  of  the  intestinal  wall  which  may  be 
found  in  the  stools,  mucus  is  of  particular  importance. 
According  to  Nothnagel,  every  admixture  of  mucus  with 
the  stools  should  be  considered  as  a  deviation  from  the 
physiological.  Mucus  may  be  seen  macroscopically  in  the 
faeces  in  varying  form,  consistency,  and  quantity. 

In  diseases  of  the  lower  portions  of  the  bowel,  mucus 
appears,  in  larger  or  smaller  quantity,  as  a  glassy  sub- 
stance, which  is  not  mixed  with  the  faeces.  In  mem- 
branous enteritis,  shreds  of  false  membrane  and  strips  of 
mucus  are  present.  When  the  mucus  comes  from  the 
upper  portion  of  the  large  intestine,  it  is  thoroughly  mixed 


FAECES  79 

with  fecal  matter  (if  the  latter  is  pasty  or  fluid  in  con- 
sistency), or  appears  in  small  strips,  just  visible  to  the 
naked  eye. 

Admixtures  of  pus,  which  can  be  recognized  macro- 
scopically,  come  from  the  lower  part  of  the  intestinal  tract, 
as  pus  coming  from  the  upper  portions  undergoes  such 
physical  and  chemical  alterations  that  its  macroscopical 
recognition  is  no  longer  possible. 

Blood  may  be  mixed  with  the  stools  in  a  fresh,  coagu- 
lated, or  decomposed  condition.  In  the  last  instance  the 
faeces  have  a  tarry  appearance.  It  is  usually  assumed  that 
the  darker  the  blood  appearing  in  the  fasces,  the  higher 
the  location  of  the  hemorrhage. 

Particles  of  tumors  (fragments  of  carcinoma,  exfoliated 
intestinal  polypi)  can  only  be  recognized  by  the  aid  of  a 
careful  histological  examination,  for  macroscopically  they 
may  be  confused  with  undigested  pieces  of  meat. 

Of  the  macroscopical  parasites  the  most  common  are : 
Proglottides  of  tapeworms,  Ascaris  lumlricoides,  Ancliy- 
lostoma  duodenale^  Oxyuris  vermicular 'is ,  TricJiocepJialus 
dispar^  and,  rarely,  insects  and  their  larvae. 

Enteroliths  and  gall-stones  are  distinguished  usually 
from  other  constituents  of  the  faeces  by  their  form,  con- 
sistency, and  surface.  They  are,  however,  not  infre- 
quently confused  by  the  patient,  as  well  as  the  physician, 
with  various  other  solid  constituents  of  the  stools ;  so  that 
a  careful  microchemical  examination  alone  renders  a  cer- 
tain determination  of  the  character  of  the  object  in  ques- 
tion possible,  in  each  individual  case. 

Foreign  bodies,  which  are  accidentally  swallowed  and 
reappear  in  the  faeces,  are  of  most  varied  character.  Usu- 
ally they  pass  through  the  intestinal  tract  unaltered,  and 
are  therefore  easily  recognized  without  further  examina- 
tion. 


80    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

5.  Quantity  of  the  Faeces. — The  daily  quantity  of  the 
faeces  differs  widely  under  normal  conditions,  and  there- 
fore no  conclusions  of  diagnostic  worth  can  be  drawn  from 
it.  The  amount  of  the  faeces  depends  upon  the  quantity 
and  character  of  the  food  and  the  condition  of  the  diges- 
tive organs.  Vegetable  foods  produce  a  much  larger 
quantity  of  faeces  than  animal.  In  pathological  conditions 
of  the  digestive  tract,  the  quantity  of  the  faeces  may  be 
markedly  increased,  due  either  to  interference  with  absorp- 
tion or  to  the  admixture  of  pathological  products  of  the 
intestinal  wall,  mucus,  pus,  blood. 

Qualitative  Chemical  Examination  of  the  Faeces 

1.  Reaction. — Under  normal  conditions  the  faeces  show 
no  marked  deviation  from  a  neutral  reaction.     They  are 
usually  faintly  alkaline  or  neutral ;  a  faintly  acid  reaction 
appears  only  following  an  exclusively  vegetable  diet.     The 
test  is  made  in  the  usual  manner  with  litmus-paper.     Two 
strips  of  litmus-paper  (red  and  blue)  are  moistened  with 
distilled  water,  applied  to  the  faeces,  and  the  change  of 
color  is  noticed  on  the  clean  side.     The  faeces  must  be 
thoroughly  mixed  before  the  examination,  as  it  frequently 
happens  that  they  are  composed  of  constituents  having 
various  reactions,  and  that  they  react  differently  on  the 
surface  than  in  the  deeper  portions.     In  addition,  the  ex- 
amination must  be  made  as  soon  as  possible  after  evacua- 
tion,  as  changes  of   reaction  often  occur  very  quickly. 
Hard  stools   must   be  thoroughly  mixed  with   distilled 
water. 

2.  Mucin. — When  the  entire  quantity  of  faeces  is  to  be 
examined  for  mucin,  they  are  thoroughly  mixed  with  water 
and  an  equal  quantity  of  lime-water  is  added  to  them. 
The  mixture  is  allowed  to  stand  for  a  few  hours,  filtered, 
and  the  filtrate  treated  with  acetic  acid.     If  mucin  is1 


FAECES  81 

present,  a  precipitate  is  thrown  down,  which  is  not  soluble 
in  an  excess  of  acetic  acid.  However,  to  recognize  the 
precipitate  with  certainty  as  mucin,  the  following  facts 
must  be  established :  ( 1 )  That  it  contains  no  phosphorus ; 
(2)  that  after  boiling  a  short  time  (ten  to  twenty  minutes) 
with  a  7.5  per  cent,  hydrochloric  acid  solution,  it  strongly 
reduces  Feliling's  solution. 

To  identify  admixtures  with  the  faeces  which  have  a 
mucous  appearance,  as  such,  by  means  of  the  detection  of 
mucin,  they  are  dissolved  in  a  weak  sodium  hydrate  solu- 
tion, and  tested  with  acetic  acid.  This  precipitate  must 
also  be  tested  for  phosphorus  and  as  to  its  reducing  power 
after  boiling  with  hydrochloric  acid. 

3.  Fat. — Fat  frequently  appears   in  the  stools  under 
normal  conditions ;  it  is  composed,  usually,  of  a  mixture 
of  neutral  fat,  fatty  acids,  and  soaps  (calcium  and  mag- 
nesium soaps). 

The  qualitative  detection  of  fat  in  the  fasces  is  very 
easy.  They  are  mixed  with  a  small  quantity  of  ether, 
allowed  to  settle,  a  small  portion  of  the  ether  withdrawn 
with  a  pipette,  and  a  drop  allowed  to  evaporate  on  a  piece 
of  filter-paper.  A  transparent  spot,  which  cannot  be 
washed  out  with  water,  remains.  The  fact  that  the  stools 
contain  fat  can,  however,  have  no  diagnostic  significance, 
since,  as  has  been  already  mentioned,  it  is  often  normally 
present  in  quantities  easy  to  detect,  particularly  following 
the  liberal  ingestion  of  fat.  Occasionally,  therefore,  for 
diagnostic  purposes,  a  quantitative  estimation  of  the  total 
fat  must  be  made. 

4.  Blood. — When  blood  in  an  undecomposed  condition 
is  mixed  with  the  faeces,  it  can  be  easily  recognized  macro- 
scopically;  as  a  control,  the  microscopical  detection  of  red 
blood-corpuscles  or  the  spectroscopical  detection  of  oxy- 
haemoglobin   is   sufficient.     When,   however,   the   blood- 


82    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

pigment  is  altered,  it  can  be  detected  only  by  chemical 
and  spectroscopical  means. 

(a}  Chemical  Detection  According  to  Weber. — A  por- 
tion of  the  faeces  is  thoroughly  mixed  with  sufficient  30 
per  cent,  acetic  acid  solution  to  render  it  liquid,  and 
extracted  in  a  test-tube  with  ether.  A  portion  of  the 
ether,  which,  when  blood  is  present,  is  brownish-red  in 
color,  is  treated  with  twenty  to  thirty  drops  of  old  turpen- 
tine and  ten  drops  of  fresh  tincture  of  guaiacum.  On 
shaking,  a  blue- violet  coloration  appears.  The  blue  pig- 
ment can,  after  the  addition  of  water,  be  extracted  with 
chloroform.  The  rest  of  the  brownish-red  ethereal  extract 
may  be  used  for — 

(b)  The  Benzidin  Test. — Put  in  a  test-tube  the  tip 
of   a  small  knifeful  of  benzidin,    1  cc  of  glacial  acetic 
acid,  and  2  cc  of  hydrogen  superoxide  and  shake  well. 
In  another  tube  rub  a  pea-sized  piece  of  faeces  with  a  glass- 
rod  in  about  5  cc  water  and  heat  to  the  boiling-point. 
Pour  a  few  drops  of  the  latter  solution  in  the  first  test-tube 
and  shake.     If  blood  is  present,  the  solution  changes  to 
from  green  to  blue-green.     This  extremely  sensitive  test  is 
of  positive  diagnostical  value  in  meat-free  diet  only. 

(c)  The  spectroscopical  examination  can  be  made  with 
the  acid-ether-extract  which  has  been  obtained  from  Weber's 
test;  this  examination  only  gives  a  positive  result,  when 
the  faeces  contain  larger  quantities  of  blood;  the  extract 
then  shows  a  distinctly  brown-red  color.     At  the  same 
time  are  seen  the  characteristic  four  absorption-bands  of 
hematin  in  acid  solution:  1.  In  red.    2.   In  yellow.    3. 
Between  yellow  and  green.  4.   Between  green  and  blue.    As 
a  rule  only  the  first  band  (in  red)  shows  distinctly. 

5.  Biliary  Constituents. 

(a)  Bile- Pigments. —  Under  normal  conditions,  the 
stools  of  the  adult  contain  no  unaltered  bile-pigment: 


FAECES  83 

bilirubin  or  biliverdin.  The  color  of  the  normal  faeces  is 
due  principally  to  the  reduced  bilirubin — hydrobilirubin 
(identical  with  urobilin). 

Hydrobilirubin  is  detected,  according  to  Schmidt,  in 
the  following  manner:  Fresh  faeces  (a  piece  the  size  of  a 
hazel-nut)  are  thoroughly  rubbed  in  a  mortar  with  a  con- 
centrated watery  solution  of  corrosive  sublimate,  and 
allowed  to  stand  for  several  hours  in  a  wide  dish.  Por- 
tions of  the  faeces  containing  hydrobilirubin  are  then  deep 
red  in  color  (due  to  the  formation  of  hydrobilirubin-mer- 
cury) ,  while  those  containing  unaltered  bilirubin  are  green. 

According  to  Schlesinger^  hydrobilirubin  in  the  faeces 
is  detected,  as  is  urobilin  in  the  urine,  by  means  of  an 
alcoholic  solution  of  zinc  acetate. 

In  addition  to  the  above-mentioned  test,  the  following 
reactions  may,  according  to  Schmidt,  be  used  to  detect 
unaltered  bilirubin  in  the  faeces. 

1.  Gmeliri's  Test. — A  few  drops  of  nitric  acid,  which 
contains  nitrous  acid,  are  placed  in  a  porcelain  dish,  and 
a  few  drops  of  faeces,  well  mixed  with  water,  are  allowed 
to  run  into  them.     A  play  of  colors  is  produced,  composed 
of  green,  blue,  violet,  red,  and  yellow.     The  green  color 
is  characteristic  of  bilirubin.     This  test  can  also  be  car- 
ried out  on  a  slide  and  observed  microscopically. 

2.  Huppertfs  Test. — Twenty  to  thirty  cc  of  faeces  are 
mixed  with  sufficient  water  to  render  them  thinly  liquid, 
treated  with  an  equal  quantity  of  milk  of  lime,   thor- 
oughly shaken  and  filtered.     The  precipitate  on  the  filter 
is  washed  with  water  and  together  with  the  filter  is  placed 
in  a  beaker,  treated  with  a  small  quantity  (5  to  10  cc)  of 
alcohol  slightly  acidified  with  sulphuric  acid,  and  carefully 
heated  to  the  boiling-point.     When  bilirubin  is  present 
the  liquid  assumes  a  green  color. 

(b)  Biliary  Adds. — Normally,  the  biliary  acids   are 


84     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

absorbed  from  the  faeces  in  the  upper  portion  of  the  in- 
testinal tract,  so  that  their  appearance  in  the  stools  must 
be  considered  as  pathological.  For  the  detection  of  biliary 
acids,  a  small  quantity  of  the  faeces  is  extracted  with 
alcohol  and  filtered.  The  filtrate  is  distilled,  to  drive  off 
the  alcohol,  and  the  residue  taken  up  by  water  rendered 
faintly  alkaline  with  soda.  Pettenkofer's  test  is  carried 
out  with  the  watery  solution;  that  is,  the  solution  is 
treated  with  cane-sugar  and  a  few  drops  of  sulphuric 
acid.  In  the  presence  of  biliary  acids  a  red  coloration  is 
produced. 

Quantitative  Chemical  Examination  of  the  Faeces 

1.  Estimation  of  Dry  Matter 

A  weighed  portion  of  the  faeces  is  first  dried  in  the  air 
on  a  water-bath.  It  is  well  to  mix  a  small  quantity  of 
dilute  sulphuric  acid  with  neutral  or  alkaline  fa3ces,  in 
order  that  there  be  no  loss  of  NH3,  which  may  be  of  im- 
portance in  a  subsequent  estimation  of  nitrogen.  The 
air-dried  faeces  are  not  yet  free  of  water,  and  must  there- 
fore be  further  dried  at  a  higher  temperature,  until  a  point 
of  constant  weight  is  reached.  This  procedure  is  difficult 
when  the  stools  are  rich  in  fat,  and  it  is  well,  therefore, 
to  evaporate  stools  containing  a  macroscopical  quantity 
of  fat  with  a  weighed  quantity  of  calcined  sand.  When 
this  is  not  done,  the  air-dried  faeces  should  be  mixed  with 
about  ten  times  as  much  weighed  sand.  Stools  not  rich 
in  fat  are  dried  in  an  air  drying-oven  at  105°  C. ;  while 
those  rich  in  fat  must  remain  about  thirty  to  forty  hours 
in  a  water  drying-oven  at  98°  to  99°  C.  The  fat  must  not 
be  subjected  to  a  higher  temperature,  as  it  melts  and  forms 
a  coating  over  the  moist  mass,  which  hinders  further  dry- 
ing. When  the  faeces  are  dried  in  an  air-oven  they  are 


F^CES  85 

weighed  every  three  hours,  until  a  point  of  constant  weight 
is  reached.  When  they  are  dried  in  a  water-oven  they  are 
first  weighed  after  twenty-four  to  thirty  hours,  and  then 
every  six  hours.  Under  a  mixed  diet  the  dry  matter  con- 
stitutes about  25  per  cent,  of  the  faeces;  under  a  purely 
vegetable  diet  it  is  considerably  less  (10  to  15  per  cent.). 

2.  Estimation  of  Total  Nitrogen 

The  nitrogen  of  the  fasces  is  usually  estimated  accord- 
ing to  the  method  of  Kjeldalil.  This  method  is  carried 
out  in  the  following  manner:  1  to  1.5  grammes  (carefully 
weighed)  of  faeces,  dried  under  the  addition  of  dilute  sul- 
phuric acid,  are  treated  in  a  Kjeldalil  flask,  with  20  cc  of 
Kjeldahl  sulphuric  acid  and  a  few  drops  of  a  concentrated 
copper  sulphate  solution,  and  allowed  to  stand  six  to 
twelve  hours.  The  flask  is  then  heated  on  a  sand-bath, 
in  a  fume-chamber,  until  the  liquid  becomes  colorless,  or 
very  faintly  wine-yellow  in  color.  Further  details  are 
carried  out  in  the  same  manner  as  in  the  estimation  of 
nitrogen  in  the  urine. 

3.  Estimation  of  Fat 

The  fat  in  the  faeces  consists  of  a  mixture  of  oleic, 
palmitic,  and  stearic  acids  and  their  salts  (soaps),  and 
glycerine  ethers  (neutral  fats).  The  relative  quantities 
of  these  components  of  the  faeces  vary  greatly,  and  depend 
principally  upon  the  character  of  the  fats  in  the  diet.  It 
is  the  estimation  of  the  total  quantity  of  fat  which  is  of 
clinical  importance;  a  separate  estimation  of  neutral  fats, 
fatty  acids,  and  soaps  is  undertaken  only  in  special  exami- 
nations, while  the  separate  estimation  of  oleic,  stearic, 
and  palmitic  acids  has  absolutely  no  practical  value. 

Estimation  of  the  Total  Fat  of  the  Faeces. — The  simplest 
method  is  extraction  with  ether.  Only  the  neutral  fats 


86     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

and  free  fatty  acids  are  soluble  in  ether.  The  soaps  must, 
therefore,  be  decomposed  before  the  extraction. 

Three  to  four  grammes  (exactly  weighed)  of  the  dried 
pulverized  faeces  are  mixed  with  a  small  quantity  of  1  per 
cent.  HC1  acid  alcohol,  and  dried  on  a  water-bath,  by 
which  procedure  the  soaps  are  decomposed.  The  dry  resi- 
due is  placed  in  the  chamber  of  a  Soxlilet  apparatus  (the 
dish  being  thoroughly  cleaned  with  pieces  of  filter-paper, 
which  are  also  placed  in  the  chamber).  Thu  extraction  is 
continued  twelve  to  twenty- four  hours.  After  the  extrac- 
tion is  completed,  the  ether,  which  is  collected  in  a  light, 
previously  weighed  flask,  is  distilled,  the  last  trace  being 
driven  off  by  a  stream  of  air,  and  the  residue  dried  for 
some  hours  at  80°  C.,  or  for  a  short  time  at  105°  C.,  and 
then  weighed. 

The  disadvantage  of  this  method  is,  that  besides  the 
neutral  fats,  fatty  acids,  and  soaps,  other  substances  solu- 
ble in  ether — as  choleeterin,  lecithin,  cholic  acid,  and 
pigments — are  included  in  the  estimation.  The  quantity 
of  these  substances  in  the  ethereal  extracts  is,  however, 
comparatively  small,  so  that  for  the  usual  clinical  estima- 
tion of  fat  it  may  be  disregarded. 

4.    Estimation  of  Carbohydrates 

(<()  Indirect  Estimation  of  Total  Carbohydrates. 

According  to  this  method  the  carbohydrates  are  esti- 
mated as  nitrogen-free  extracts,  the  values  of  the  albumin, 
fat,  and  ash  being  subtracted  from  the  dried  fecal  matter. 
It  is  self-evident  that  this  method  gives  comparatively 
inexact  and  practically  useless  results :  first,  because  the 
estimated  residue  contains  other  substances  in  addition 
to  carbohydrates  (vegetable  acids,  pigments,  etc.);  sec- 
ondly, because  no  judgment  of  the  extent  of  digestion  can 
be  formed  from  the  total  quantity  of  carbohydrates. 


FAECES  87 

Such  a  judgment  can  only  be  rendered  possible  by  sep- 
arating the  practically  indigestible  cellulose  from  the  read- 
ily soluble  starch.  As,  however,  we  possess  no  exact  and 
simple  method  for  the  quantitative  estimation  of  cellulose, 
a  direct  estimation  of  starch  is  undertaken  in  order  to 
judge  of  the  extent  of  the  digestion  of  the  carbohy- 
drates. 

(/O  Direct  Estimation  of  Starch  According  to  Liebermann 
and  Allihn. 

The  principle  of  this  procedure  is  that  starch  is  con- 
verted into  grape-sugar  by  boiling  with  hydrochloric  acid, 
the  sugar  solution  boiled  with  Fehlimfs  solution,  and  the 
precipitated  copper  oxide  reduced  by  hydrogen  to  metallic 
copper.  From  the  quantity  of  copper  the  quantity  of 
grape-sugar  is  determined,  and  from  it  the  starch  is  cal- 
culated. 

When  the  faeces  contain  a  liberal  admixture  of  mucus, 
it  must,  as  far  as  possible,  be  removed  with  forceps,  as 
mucin,  when  boiled  with  hydrochloric  acid  forms  a  copper 
reducing  substance. 

Three  to  five  grammes  of  dried,  pulverized,  and  exactly 
weighed  faeces  are  treated  in  a  flask  with  100  cc  of  a  2  per 
cent,  solution  of  hydrochloric  acid,  and  boiled  on  a  sand- 
bath  for  an  hour  and  a  half,  using  a  back-flow  condenser; 
the  liquid  is  then  neutralized  with  sodium  hydrate,  and 
filtered  through  an  asbestos  filter,  by  means  of  an  exhaust- 
pump,  into  a  500  cc  flask,  and  the  residue  washed  with 
hot  water  until  the  filtrate  amounts  to  500  cc.  Thirty  cc 
of  a  7  per  cent,  solution  of  copper  sulphate  (Fehling^s 
solution  No.  1),  80  cc  of  an  alkaline  solution  of  Rochelle 
salts  (Fehling's  solution  No.  2),  and  60  cc  of  water  are 
placed  in  a  beaker  or  porcelain  dish  and  heated  to  the 
boiling-point.  To  the  boiling  liquid  25  cc  of  the  sugar 
solution  are  added  from  a  pipette,  the  liquid  boiled  three 


88    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

minutes,  and  the  precipitated  copper  oxide  collected  on  a 
filter. 

For  filtering,  an  asbestos  filter-tube  is  used.  The  tube 
must  be  filled  with  long- fibred,  soft  asbestos;  to  prevent 
particles  of  asbestos  from  being  washed  through  during 
the  infiltration,  a  small  plug  of  glass  wool  is  placed  at  the 
conical  end  of  the  tube  underneath  the  asbestos.  The  fil- 
tration is  best  accomplished  by  means  of  an  exhaust- 
pump.  The  tube  is  dried  at  100°  C.,  and  weighed  before 
using.  The  copper  oxide  collected  on  the  asbestos  filter 
is  first  washed  with  cold  water,  then  with  alcohol  and 
ether,  and  finally  dried  for  fifteen  minutes  in  a  drying- 
oven  at  100°  C.  A  stream  of  pure,  dry  hydrogen  gas  is 
now,  under  slight  heating,  allowed  to  flow  from  a  Kipp*s 
hydrogen  generator  through  the  dry  tube.  As  soon  as  the 
precipitate  has  assumed  the  characteristic  copper  color, 
and  the  tube  is  thoroughly  dry,  the  heating  is  stopped, 
the  tube  is  allowed  to  cool  in  the  stream  of  hydrogen,  and 
weighed.  From  the  amount  of  copper  oxide  found  the 
amount  of  grape-sugar  is  calculated. 

(c)  Fermentation  Test  According  to  Schmidt. 

This  test  renders  possible  the  detection  and  approxi- 
mate quantitative  estimation  of  the  carbohydrates,  which 
are  easily  acted  upon  by  the  digestive  juices,  and  is  there- 
fore, especially  as  its  performance  is  very  simple,  to  be 
recommended  as  a  method  for  estimating  the  efficiency  of 
the  digestive  apparatus. 

The  principle  of  this  method  is  that  the  dissolved  car- 
bohydrates, as  well  as  those  starches  which  lie  free  and 
are  easily  acted  upon  (enclosed  in  thin  cellulose  capsules), 
are  inverted  by  the  diastase  which  is  always  present  in  the 
faeces,  and  are  then  fermented  by  the  intestinal  bacteria, 
with  the  production  of  gas.  The  test  is  carried  out  in  the 
following  manner :  5  grammes  of  f a3ces  are  placed  in  the 


FAECES 


89 


vessel  of  a  Schmidt's  fermentation  apparatus  (Fig.  12), 
well  mixed  with  water,  and  the  vessel  closed  with  the  rub- 
ber stopper,  care  being  taken  to  exclude  air-bubbles. 
Tube  I  is  also  filled  with  water,  without  air-bubbles  and 


FIG.  12 

closed  with  the  smaller  rubber  stopper.  The  entire  appar- 
atus is  then  placed  in  an  incubator  (37°  C. )  for  twenty- 
four  hours.  The  gas  which  is  developed  by  the  fermenta- 
tion forces  a  portion  of  the  water  from  tube  b  into  tube  c. 


90     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

The  air  in  tube  c  escapes  through  the  opening  d.  The 
quantity  of  gas  produced,  which  corresponds  to  the  quan- 
tity of  fermentable  carbohydrates,  is  judged  from  the 
height  of  the  water  in  tube  c.  For  diagnostic  purposes 
only  a  positive  result  of  the  test  is  of  value,  as  under 
pathological  conditions  the  test  may  be  negative  even 
when  sugar  and  starch  are  present. 

According  to  Schmidt^  intestinal  dyspepsia  may  be 
diagnosed  when,  following  a  test-diet  suggested  by  him- 
self and  Strassburger,  enough  gas  is  formed  in  twenty- 
four  hours  to  fill  tube  c  at  least  one- fourth  full  of  water. 

The  test-diet  consists  of — 

1.5  litres  of  milk 

3J  eggs. 

Gruel  from  80  grammes  of  oatmeal. 

100  grammes  of  zwieback  (rusk). 

20  grammes  of  sugar. 

20  grammes  of  butter. 

125  grammes  of  beef       ) 

190  grammes  of  potato   ' 


raw. 


Examination  of  Gall-Stones  and  Biliary  Concretions 

General  Characteristics 

Gall-stones  are  usually  pale  yellow,  or,  more  rarely, 
brownish-red  in  color.  Stones  of  pure  cholesterin  are 
nearly  colorless,  and  show  a  distinctly  crystalline  charac- 
ter. Their  size  varies  greatly,  from  that  of  a  pin's  head 
to  that  of  a  walnut.  They  vary  in  hardness,  though,  as 
a  rule,  they  are  much  softer  and  lighter  than  typical 
enteroliths. 

On  cross-section,  gall-stones  show  not  infrequently  a 
distinct  nucleus  and  a  marked  concentric  stratification. 


FAECES  91 

Xaunyn  divides  gall-stones,  according  to  their  chemi- 
cal characteristics,  into  the  following  groups: 

1.  Pure  cholesterin  stones  with  smooth  or  warty  sur- 
face :  on  section  white,  and  of  crystalline  structure. 

2.  Stratified  cholesterin  stones :  colored  and  stratified. 

3.  Ordinary  gall-stones:  stratified,    colored,   but  not 
crystalline. 

4.  Mixed    bilirubin-calcium    stones:    stratified   and 
colored,  the  nucleus  consisting  usually  of  cholesterin. 

5.  Pure  bilirubin-calcium  stones:  dark  brownish-red 
in  color,  the  principal  constituents  being  combinations  of 
calcium  with  the  biliary  pigments — bilirubin,  biliverdin, 
bilifuscin,  and  biliprasin,  cholesterin  being  present  in  very 
small  quantity,  or  not  at  all. 

6.  Amorphous  cholesterin  stones,  conglomerate  stones, 
and  casts  of  the  biliary  passages,  which  are  very  rare. 

Chemical   Examination 

As  gall-stones  and  biliary  concretions  are  combinations 
of  calcium  with  biliary  pigments,  it  is  necessary  for  their 
identification  as  gall-stones,  in  cases  in  which  the  nature 
of  the  stones  is  not  known,  to  detect  chemically  these 
principal  constituents.  For  this  purpose  the  following 
procedure  is  carried  out:  A  stone  is  pulverized  and  boiled 
in  water.  By  this  means  any  traces  of  biliary  acids  which 
may  be  present  are  removed.  The  residue  is  then  extracted 
with  a  warm  mixture  of  equal  parts  alcohol  and  ether. 
The  cholesterin  is  dissolved;  the  residue  (1)  contains  the 
bile-pigments,  which  are  combined  with  calcium,  and  the 
inorganic  salts,  which  are  insoluble  in  water.  For  the 
detection  of  cholesterin  the  alcohol  and  ether  solution  is 
separated  from  the  residue  by  centrifugalization,  and  is 
allowed  to  evaporate.  When  cholesterin  is  present  it 
forms  large,  very  thin,  characteristically  placed,  colorless, 


92     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

rhomboid  plates;   more  rarely,    it  forms  needles  with  a 
silky  lustre. 

For  the  identification  of  cholesterin  the  following  re- 
actions are  used : 

1.  Concentrated  sulphuric  acid  is  allowed  to  run  into 
the  cholesterin  on  a  slide,  the  crystals  then  dissolve  at  the 
edges  and  assume  a  carmine  color;   if  LugoVs  solution  is 
added,  a  blue,   red,  green,   and  violet  play  of  colors  is 
seen. 

2.  A  small  quantity  of  perfectly  dry  cholesterin  is  dis- 
solved in  glacial  acetic  acid,  and  a  few  drops  of  concen- 
trated sulphuric  acid  are  added;    a  violet  coloration  is 
produced,  which  very  quickly  becomes  green.     The  test 
succeeds  only  when  the  cholesterin  is  absolutely  dry. 

For  the  detection  of  bilirubin-calcium  the  residue 
(1)  is  covered  with  hydrochloric  acid  (when  calcium  car- 
bonate is  present  foam  is  produced),  and  heated.  The 
biliary  pigments  are  by  this  means  freed  from  their  union 
with  calcium.  After  cooling,  the  bilirubin  is  extracted 
with  chloroform.  The  chloroform-extract  may  then  either 
be  allowed  to  crystallize,  or  may  be  used  in  carrying  out 
Gmelin's  test. 

Fecal  Concretions,  Enteroliths,  and  Pancreatic 
Stones 

By  fecal  concretions  or  coproliths  are  meant  stony 
bodies,  which  are  composed  of  hardened  fecal  matter. 
They  are  formed,  as  a  rule,  in  those  places  in  the  large 
intestine  at  which  stagnation  of  fecal  matter  can  most 
easily  take  place;  for  example,  at  the  flexures,  or  in  the 
appendix  vermiformis.  Coproliths  may  reach  such  size 
and  compactness  that  they  cause  complete  intestinal  ob- 
struction. True  intestinal  stones  (enteroliths)  are  much 
smaller  than  fecal  concretions,  and  have  in  their  entire 


FAECES  93 

character  a  much  closer  resemblance  to  other  kinds  of 
stones  (urinary  and  biliary  calculi ) .  They  consist  usually 
of  a  nucleus  of  organic  matter  (fruit-pips,  blood-clots, 
particles  of  faeces,  etc.),  about  which  layers  of  salts  (usu- 
ally earthy  or  triple  phosphates)  have  been  deposited.  A 
distinction  is  made  between  the  following  forms  of  entero- 
liths : 

1.  Typical  Enteroliths. — These  are  round,  heavy,  stone- 
hard,    concentrically    stratified,     and  contain    a   foreign 
body,  the  nucleus  of  the  concrement. 

2.  Light  Stones. — These   are   composed   principally  of 
undigested  vegetable  food  particles,  encrusted  with  phos- 
phates.    They  are  not  stratified,    and  have  no    distinct 
nucleus.     To  this  group  belong  the  so-called  "oatmeal- 
stones,"  which  may  form  after  the  liberal  and  prolonged 
ingestion  of  oatmeal. 

3.  Stones  Composed  of  Drugs  which  have  been  Taken. — 
Such  stones  consist  principally  of  insoluble,  or  difficultly 
soluble  drugs,  which  were  taken  in  powder  form.     For 
example,  salol,  magnesia,  calcium  carbonate,  etc. 

4.  Intestinal  Gravel. — This  consists  of  small,  hard  gran- 
ules, which  are  usually  composed  of  organic  matter,  cal- 
cium carbonate,  and  magnesium  phosphate. 

5.  Pancreatic  Stones. — These  are  very  rarely  found  in 
the  faeces.    They  are  crumbly,  and  have  a  rough  surface. 
They  are  readily  soluble  in  chloroform,  and  on  heating 
give  off  an  aromatic  odor.     They  are  usually  composed 
of  calcium  carbonate  and  phosphate.     In  the  few  cases 
reported  in  literature  cholesterin  and  bile-pigments  were 
detected. 

For  examination,  the  calculi  are  sawn  through,  and  a 
small  piece  pulverized  and  tested  by  burning  on  a  plati- 
num spatula.  If  most  of  the  powder  burns  up,  the  cal- 
culus consists  principally  of  organic  substances.  In  such 


94     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

cases  microscopical  examination  will,  in  the  majority  of 
instances,  disclose  the  composition  of  the  calculus. 

If,  on  the  contrary,  the  calculus  merely  turns  black  on 
burning,  leaving  considerable  residue,  it  is  composed  prin- 
cipally of  inorganic  substances.  A  qualitative  analysis 
of  these  substances  is  carried  out  in  the  following  manner : 
A  portion  of  the  pulverized  stone  is  treated  in  a  test-tube 
with  dilute  hydrochloric  acid,  and  slightly  heated.  If  gas 
develops  on  the  addition  of  the  hydrochloric  acid,  carbon- 
ates are  present.  The  portions  insoluble  in  hydrochloric 
acid  consist  principally  of  sand  or  of  organic  matter,  and 
must  be  examined  microscopically,  the  hydrochloric  acid 
solution  being  separated  from  the  residue  either  by 
filtration  or  centrifugalization.  The  fluid  may  contain 
phosphates  (of  calcium  magnesium),  calcium  oxalate, 
ammonia,  and  traces  of  albuminoid  substances.  The  de- 
tection of  these  constituents  is  accomplished  in  the  same 
manner  as  in  the  examination  of  urinary  calculi  (q.v.). 

Microscopical  Examination  of  the  Faeces 

Fluid  or  thin,  pasty  stools  are  poured  into  a  shallow 
dish,  and  when  they  are  uniform  in  consistency  a  small 
portion  is  taken  and  spread  between  a  cover-glass  and  slide. 
Any  macroscopical  objects  which  attract  attention  must 
be  examined  separately.  Very  thin  stools  are  allowed  to 
settle,  or  are  centrifugalized,  and  the  sediment  examined. 
Formed  stools  are  rubbed  in  a  glass  mortar  with  water  or 
physiological  salt  solution.  During  the  microscopical  ex- 
amination principally  food  particles  (the  great  majority 
of  which  are  of  vegetable  origin),  bacteria,  and  crystal- 
line bodies  in  small  number  are  found.  Under  patholo- 
gical conditions,  pathological  products  of  the  intestinal 
wall  and  animal  parasites  may  be  present. 

Of  the  food  particles  only  those  will  be  considered  here 


FAECES  95 

whose  presence  in  the  stools  may  be  of  diagnostic  signifi- 
cance.    Among  these  are  included : 

1.  Muscle- Fibres. — These  are  in  the  stools  nearly  always 
heavily  stained  by  bile-pigments,  and  are  therefore  easy 
to  find.     They  are  divided  by  Schmidt,  according  to  their 
form  and  structure,  into  three  groups : 

(«)  Large. — Distinctly  striated  pieces  with  sharp  cor- 
ners and  outline. 

(b)  Medium. — Rectangles  with  rounded  corners,  whose 
striae  are  still  visible. 

(c)  Small. — Polygonal  or  round  flakes,  mostly  homo- 
geneous and  with  indistinct  striae. 

The  presence  in  the  stools,  following  the  limited  inges- 
tion  of  meat,  of  numerous  muscle-fibres  indicates  a  dis- 
turbance of  the  function  of  the  small  intestine,  probably 
of  the  pancreatic  digestion.  When  the  food  has  been 
insufficiently  masticated,  shreddy  objects,  consisting 
principally  of  half-digested  meat  particles,  may  be  fre- 
quently seen  macroscopically  in  the  stools  of  healthy 
persons. 

2.  Shreds  of  connective  tissue  are  frequently  seen  during 
the  macroscopical  examination  of  the  faeces.     On  micro- 
scopical examination  they  show  a  thready  structure  with 
delicate,  often  scarcely  recognizable,  fibrillation.     In  cer- 
tain portions  the  interwoven  elastic  fibres  can  be  distinctly 
seen.     Upon  the  addition  of  acetic  acid  the  structure  of 
the  connective  tissue  disappears,  while  the  elastic  fibres 
become  more  distinct. 

The  presence  of  much  connective  tissue  in  the  stools, 
following  the  limited  ingestion  of  meat  (100  grammes), 
points  toward  disturbance  of  the  gastric  digestion,  since 
the  gastric  juice  alone  is  able  to  dissolve  raw  or  incom- 
pletely cooked  connective  tissue.  Following  the  ingestion 
of  smoked  meats  the  presence  of  connective  tissue  in  the 


96     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

stools  may  be  considered  normal,  as  such  raw  connective 
tissue  is  digested  with  tjie  greatest  difficulty. 

3.  Occasional  starch  granules   appear  even  in  normal 
stools.     Their  marked  increase  indicates  disturbance  in 
the  small  intestine. 

4.  Fat- — Fat  is  present  in  small  quantity  in  all  stools, 
and  appears  in  the  form  of  drops,  flakes  (neutral  fat),  or 
crystals    (fatty  acids,    soaps).     Fatty   acids    are   distin- 
guished   from  soaps    by  the  fact  that  they  melt   when 
heated,  while  soaps  remain  unchanged.     In  addition,  fatty 
acids  dissolve  readily  in  ether,  while  soaps  must  first  be 
decomposed  by  acids.     Upon  the  addition  of  a  saturated 
alcoholic  solution  of  Sudan  III.,  neutral  fat  assumes  an 
orange  to  blood-red  color,   while  fatty    acids  and  soaps 
remain  colorless.     Fat  is  increased  in  the  stools   in  all 
diseased   conditions    in   which   there    is  an  interference 
with    its    absorption    from   the    food   (affections   of    the 
intestinal   mucosa,    interference   with   biliary   secretion, 
etc.). 

In  addition  to  the  above-mentioned  fatty  acid  crystals, 
the  following  crystalline  bodies  may  be  seen  during  the 
microscopical  examination  of  the  faeces :  Triple  phosphate 
("coffin-lid"),  neutral  calcium  phosphate,  magnesium 
phosphate,  calcium  oxalate  ("envelope"),  calcium  car- 
bonate, calcium  sulphate,  cholesterin,  and  Char  cot- Ley  den 
crystals  (in  helminthiasis  and  enteritis  membranacea). 

The  Pathological  Products  of  the  Intestinal  Wall  Espe- 
cially to  be  Considered  are : 

1.  Mucus- — This  appears  microscopically  as  a  struct- 
ureless, transparent  mass,  in  which  epithelial  cells, 
pus-corpuscles,  crystals,  or  food  particles  are  frequently 
embedded.  Upon  the  addition  of  acetic  acid  (the  mucus 
fleck  should  be  thoroughly  mixed  with  the  reagent),  the 
basic  substance  assumes  a  striated  appearance.  The  pres- 


FAECES  97 

ence  of  mucus  in  the  fasces  nearly  always  indicates  a  patho- 
logical condition  of  the  intestinal  mucosa. 

2.  Epithelium- — Squamous  cells  are  very  rarely  present 
in  the  stools  (diseases  of  the  rectum) ;  cylindrical  cells, 
however,  are  more  frequent.  They  rarely  appear  unchanged, 
but  frequently  in  the  so-called  "verschollter"  form  (des- 
quamated lumps),  or  in  a  half-digested  condition.     The 
presence  of  small  mucus    shreds,   containing  only  half- 
digested  epithelium,  indicates  inflammation  of  the  small 
intestine.     Desquamated  epithelium  comes  usually  from 
the  large  intestine.     The  presence  of  a  large  amount  of 
epithelium  in  the  stools  indicates  usually  a  catarrhal  in- 
flammation of  the  intestinal  mucosa. 

3.  Pus- Corpuscles- — Leucocytes  in  small  numbers  are 
found  in  every  mucus  fleck.     The  appearance  of  a  great 
number  of  pus-corpuscles  indicates  an  ulcerative  process 
in  the  intestines. 

Red  blood-corpuscles  appear  in  the  stools  in  unaltered 
condition  only  when  the  blood  comes  from  the  lower  por- 
tions of  the  intestines,  and  has  remained  in  them  but  a 
short  time.  If  the  blood  comes  from  the  upper  portions 
of  the  intestines,  the  so-called  "shadow  corpuscles"  may 
be  occasionally  found.  As  a  rule,  however,  red  blood- 
corpuscles  can  no  longer  be  detected. 

Intestinal  Parasites  and  Their  Eggs 

1.  Amoebae — According  to  Quincke  and  Roos,  three 
kinds  of  amoeba?  are  parasitic  in  man:  Ammba  vulgaris, 
mitis,  and  coli  (dysentery).  Recently  the  first  two  have 
been  considered  as  one.  The  Amoeba  coli  alone  is  accred- 
ited with  pathological  significance  (Fig.  13).  It  is  10  to 
15  millimetres  in  length,  and  is  very  motile.  It  contains, 
in  addition  to  bacteria  and  ingesta,  red  blood-corpuscles, 
which  is  never  the  case  with  the  Amoeba  vulgar  is  or  mitis. 


98     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Amwba  coli  is  considered  by  most  authors  as  the  exciting 
cause  of  amoebic  dysentery.  Its  encysted  form  has  a 
simple  contour,  while  the  encysted  forms  of  the  other  two 
varieties  have  a  double  contour. 


FIG.  13. — Amoeba  Coli  (a,  according  to  Roemer  ;  b,  according 
to  Doflein) 

The  examination  of  the  faeces  for  amoebae  must  be  made 
as  soon  as  possible  after  evacuation,  as  these  parasites  re- 
sist cooling  very  feebly,  and  disappear  rapidly.  When 
the  faeces  are  not  perfectly  fresh,  only  the  encysted  forms 
are  to  be  found. 

2.  Infusoria  (Fig.  14). — These  are  enclosed  in  a  hard 
capsule,  the  surface  of  which  is  covered  with  flagella  or 


FIG.  14. — Balantidium  Coli  (according  to  Leuckart) 

cilia.     In  the  faeces  are  found:   Cercomonas  intcstinaKs, 

Tricliomonas    intestinalis,   and    Balantidium  coli.      The 
last  only  is  accredited  with  pathological  significance.     It 


FAECES 


99 


is  not  infrequently  found  in  intestinal  ulcerations.  Whe- 
ther this  parasite  enters  the  ulcerated  mucosa  seconda- 
rily, or  is  the  cause  of  the  ulcerative  process,  is  not  yet 
proved.  The  majority  of  authors  doubt  whether  it  is 
pathogenic  for  man. 

8.   Tapeworms  (Cestodes). 

(a)  Tcenia,  Solium  (Fig.  15).  —  The  cysticercus  lives 
in  swine.  The  worm  is  2  to  3  millimetres  long.  Its 
scolex  is  unpigmented,  has  four  suckers  and  a  rostellum, 
which  carries  a  double  crown  composed  of  twenty-six 


FIG.  15.— Tsenia  Solium :  Scolex,  Proglottides,  Egg. 
(After  v.  Jaksch. ) 

booklets.  The  ripe  segments  (proglottides)  are  rather 
long  when  shed.  The  uterus  has  but  seven  to  ten  branches. 
The  eggs  are  usually  round  (rarely  oval),  and  enclosed  in 
a  thick  shell,  in  which  a  distinct  radial  striation  is  seen. 
Not  infrequently  the  booklets  of  the  embryo  are  visible 
within  the  egg. 

(#)  Tcenia  Saginata  (inediocanellatci)  (Fig.  16). — The 
cysticercus  lives  in  the  muscles  of  the  ox.  The  worm  is  4 
to  8  millimetres  long.  Its  scolex  has  no  rostellum  and 


100     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 


no  crown  of  booklets;  it  has  four  pigmented  suckers. 
The  uterus  has  twenty  to  thirty  branches.  These  are  best 
seen  when  the  ripe  segment  is  squeezed  between  two  slides. 


FIG.  16.— Taenia  Saginata:  Scolex,  Eggs,  Proglottides. 
(After  v.  Jaksch.) 

The  eggs  are  somewhat  larger  than  those  of  the  Tcenia 
solium,  but  are  in  other  respects  hard  to  distinguish  from 
them. 

(c)  Bothriocephalus  Latus  (Fig.  17). — The  cysticercus 
lives  in  salt-  and  fresh- water  fish.  The  worm  is  6  to  8 
millimetres  long.  Its  long  scolex  with  its  long  neck  is 


a  b  c 

FIG.  17. — Scolex  of  Bothriocephalus  Latus.     a,  Seen  from 
above ;  b,  from  the  side ;  c,  proglottides ;  d,  eggs. 
(After  v.  Jaksch.) 


F^CES 


101 


flattened  out,  and  has  two  elongated  suckers.  The  eggs  are 
oval,  and  have  a  lid  at  one  end.  When  the  embryo  is 
discharged  the  lid  is  lifted.  The  ripe  segments  are  quad- 
rilateral, and  show  a  rosette  marking  in  the  centre,  due 
to  the  brown  egg- filled  uterus. 

The  following  tsenise  are  more  rarely  seen:  Tcenia 
nana,  Tcenia  flavopunctata,  and  Tcenia  cucumerina. 
Tcenia  nana  is  common  in  Italy  and  Egypt. 

4.  Round  Worms  (Nematodes). 

(a)  Oxyuris  Vermicularis  (Fig.  18). — The  eggs  of 
this  worm  are  swallowed  and  pass  into  the  faeces,  in  which 

c  b  a 


FIG.  18. — Oxyuris  Vermicularis.     a,  Scolex  ;  b,  female; 
c,  male  worm ;  d,  eggs.     (After  v.  Jaksch. ) 

the  worm  completes  its  development.  The  male  is  4 
millimetres,  the  female  10  millimetres  long.  The  eggs 
have  a  double  contour,  and  are  usually  filled  with  a 
coarsely  granular  substance.  Occasionally  an  egg  is  seen 


102     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 


containing  an  embryo,  in  which  the  intestinal  canal  can 
be  indistinctly  seen. 

(#)  Ascaris  Lumbricoides  (round  ivorm)  (Fig.  19). — 
This  worm  is  cylindrical  and  comparatively  long — 20  to 


FlG.  19. — Ascaris  Lumbrico- 
ides.  a,  Scolex ;  6,  caudal  ex- 
tremity of  the  male  worm; 
c,  egg ;  d,  male  worm. 

(After  v.  Jaksch. ) 


FIG.  20.— Trichocephalus  Dis- 
par.  a,  Male ;  6,  female 
worm ;  c,  egg. 


40  centimetres.     The  eggs  are  round  or  oval,  yellowish- 
brown  in  color,  and  enclosed  in  an  albuminoid  capsule. 

(c)   Triclioceplialus  Dispar  (whip-worm)  (Fig.  20).— 
This  is  usually  considered  as  a  harmless  intestinal  para- 


FAECES 


103 


site;  recently,  however,  Metsclmikoff  has  accredited  to  it  a 
significance  in  inflammation  of  the  appendix  vermiformis. 
The  worm  is  about  4  centimetres  in  length.  The  eggs  are 
easily  recognized  by  the  lid  which  they  have  at  either  end. 
They  have  a  double  contour,  are  brownish  in  color,  and 
are  filled  with  a  granular  substance. 

(d)  Ancliylostomum  Duodenale  (Fig.  21). — As  a  rule, 
only  the  eggs  are  found  in  the  faeces,   since  the  worms 


FIG.  21. — Anchylostoma   Duodenale.     a,  Male   worm    (natural 
size) ;  b,  female  worm  (natural  size)  ;  c,  male  worm  (slight- 
ly  magnified)  ;  d,   female  worm    (slightly  magnified)  ;   e, 
scolex ;  /,  eggs ;  g,  caudal  extremity  of  the  male. 
(After  v.  Jaksch. ) 


themselves  are  so  deeply  and  firmly  embedded  in  the  in- 
testinal wall  (small  intestine)  that  they  are  not  evacuated 
with  the  stools.  The  eggs  have  a  single  contour,  are  oval, 
and  contain  all  stages  of  development  of  the  embryo  side 
by  side.  The  male  is  10  millimetres  long,  and  has  two 


104     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

spicules  at  its  caudal  extremity.     The  female  is  pointed 
at  the  caudal  extremity,  and  is  12  to  18  millimetres  long. 

Bacteriological  Examination  of  the  Faeces 

The  f seces  possess  normally  a  very  luxuriant  bacterial 
flora,  the  many  varieties  of  which  can  be  distinctly  seen 
in  specimens  stained  with  dilute  carbol-f  uchsin  and  accord- 
ing to  Gram.  The  bacteria  present  in  greatest  number 
are  those  belonging  to  the  group  of  Bacterium  coli.  In 
addition,  Bacillus  aeroyenes,  varieties  of  subtilis  and 
proteus,  B.  fcecalis  alkaligenes,  B.  fluorescens,  different 
varieties  of  cocci,  fungi,  and  yeast-cells,  are  present.  In 
cultures  from  the  faeces  only  a  very  small  per  cent,  of  these 
micro-organioms  (about  10  per  cent. )  develop.  Upon  the 
usual  culture  media  bacteria  of  the  coli  group  grow  in 
overwhelming  majority. 

The  most  important  pathogenic  bacteria  found  in  the 
faeces  are  typhoid,  cholera,  dysentery,  and  tubercle  bacilli, 
more  rarely  strepto-  and  staphylococci,  anthrax  bacilli, 
plague  bacilli,  and  B.  pyocyaneus. 

Typhoid  Bacilli 

The  detection  of  typhoid  bacilli  in  the  faeces  is  still 
attended  with  considerable  difficulty,  and  may  not  succeed 
even  in  cases  which  manifest  themselves  clinically,  as  un- 
doubted typhoid.  Often  only  repeated  and  laborious 
attempts  succeed.  Attempts  made  with  the  typical  diar- 
rhceal  evacuations  are  the  most  likely  to  be  successful, 
either  because  the  bacteria  are  discharged  in  greater  num- 
bers or  are  more  evenly  distributed  than  in  formed  stools. 
In  the  latter  they  are  frequently  present  in  isolated  spots 
only,  in  which  case  the  presence  of  any  bacteria  at  all  in 
the  material  used  for  inoculation  is  more  or  less  a  matter 
of  chance. 


FAECES  105 

Characteristics  of  Typhoid  Bacilli. — Morphological  and 
Staining  Characteristics. — The  typhoid  bacillus  is  a  short 
rod  which  stains  easily  with  dilute  aniline  dyes,  and 
is  decolorized  by  Gram.  In  hanging  drops,  typhoid 
bacilli,  when  grown  on  suitable  culture  media,  are  very 
motile. 

Growth  on  the  Usual  Culture  Media. — Typhoid  bacilli 
grow  upon  all  the  usual  culture  media,  and  best  at  body- 
temperature. 

Upon  agar  they  develop  small,  moist,  grayish-white 
colonies,  which,  when  held  against  the  light,  show  a  blu- 
ish iridescence.  They  are  more  delicate,  smaller,  and 
more  transparent  than  those  of  the  B.  coli  communis. 

On  gelatine  the  surface  colonies  have  usually  a  charac- 
teristic appearance;  they  appear  delicate,  iridescent,  with 
jagged  or  wavy  margins,  and  are  traversed  by  numerous 
branching  ridges  resembling  the  ribs  of  a  grape-leaf 
(grape-leaf  form).  This  growth  is,  however,  in  no  wise 
typical  of  typhoid  bacilli  alone,  for  there  are  varieties  of 
coli  whose  colonies  present  the  same,  or  a  very  similar, 
appearance.  Typhoid  bacilli  do  not  liquefy  gelatine. 

On  potato  they  develop  a  fine,  colorless  coating,  invis- 
ible to  the  naked  eye.  There  are,  however,  varieties  of 
potato  upon  which,  especially  in  the  presence  of  an  alka- 
line reaction,  a  gray,  slimy  coating  is  produced. 

Bouillon  is  evenly  clouded. 

Groivtli  of  Typlioid  Bacilli  on  Special  Culture  Media. — 
In  the  endeavor  to  simplify  the  isolation  of  typhoid  bacilli 
from  mixtures  of  bacteria,  especially  in  attempts  to  culti- 
vate from  the  faeces,  a  number  of  culture  media  have  been 
suggested,  upon  which  typhoid  bacilli  show  conspicuous 
differences  in  their  growth  from  other  bacteria,  particularly 
from  those  of  the  coli  group.  Only  the  two  culture  media 
most  frequently  used  will  be  mentioned  here :  the  Conradi- 


106     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Drigalslci  litmus-lactose-agar,  and  the  PiorlcowsM  urine- 
gelatine  (for  the  preparation  of  these  culture  media,  cf. 
Chapter  XII). 

Upon  the  Conradi-Drigalslci  culture  medium  typhoid 
bacilli  produce,  after  fourteen  to  twenty- four  hours'  growth 
at  87°  C. ,  small  glassy  colonies  with  single  contour,  resem- 
bling dew-drops,  and  bluish  in  color,  with  a  tinge  of  vio- 
let. Earely,  however,  the  larger  colonies  have  a  more 
clouded  appearance.  The  colonies  of  B.  coli  are  larger 
than  those  of  typhoid  bacilli,  and  are  usually  brilliant  red 
and  non-transparent.  l  i  Many  colonies  are  only  bright  red, 
and  not  so  cloudy;  other  varieties  of  coli  produce  larger 
colonies  of  waxy  appearance,  which  are  surrounded  by  a 
red-stained  area. ' ' 

It  must,  however,  be  mentioned  that  there  are  other 
bacteria  whose  growth  does  not  change  the  color  of  this  cul- 
ture medium.  Their  colonies  are  frequently  distinguished, 
however,  from  those  of  typhoid  bacilli  by  their  size, 
distinctly  double  contour,  and  their  dull,  dry  surface. 
Among  these  are  B.  fcecalis  alkaligenes,  and  bacteria  of 
the  subtilis,  proteus,  and  fluorescens  groups.  The  colonies 
of  streptococci,  which  in  attempts  at  cultivation  from 
the  fasces  often  develop  in  great  numbers  on  this  culture 
medium,  also  exactly  resemble  in  color  the  colonies  of 
typhoid  bacilli.  They  are,  however,  very  much  smaller 
than  these. 

In  endos  fuchsinagar  after  having  grown  for  twelve 
hours  at  87°  the  coli-colonies  become  gradually  red,  starting 
from  the  centre ;  after  twenty- four  hours  they  are  entirely 
red,  round  with  prominent  edges.  The  typhoid  bacilli  on 
the  other  hand  are  forming  colorless  round  colonies 
with  thin  edges.  After  more  than  twenty-four  hours  the 
coli-colonies  are  dark-red,  while  the  typhoid-colonies — now 
twice  the  size  of  the  coli-colonies — remain  colorless  or  are 


FAECES  107 

of  a  faint  reddish  color.  The  colonies  of  the  bacteria 
which  grow  blue  in  the  Conradi-Drigalski  culture  medium 
look  like  typhoid-colonies  in  fuchsin-agar. 

This  culture  medium  offers  advantages  against  the  lit- 
mus-lactose-agar,  because  it  is  prepared  much  easier  and 
one  can  work  in  artificial  light,  while  the  blue  colonies 
on  the  DrigalsM-platea  can  be  recognized  in  daylight  only. 
A  great  disadvantage  is  the  fact  that,  in  endo-agar  with 
many  acid-formers  present,  the  culture  medium  becomes 
diffusely  red,  which  renders  it  impossible  to  recognize  the 
colorless  typhoid-colonies. 

Thus  the  diagnosis  cannot  be  made  from  the  appear- 
ance of  the  colony  alone,  if  fuchsin-agar  and  Conradi- 
Drigalski  culture  medium  have  been  used ;  the  suspicious 
looking  bacteria  have  to  be  tested  as  to  their  morphological 
and  biological  properties  in  the  same  way  as  they  would 
have  been  cultivated  in  ordinary  agar.  The  fuchsin-agar 
and  the  litmus-lactose-agar  offer  the  advantage  over  the 
agar,  that  they  make  it  easier  to  detect  the  suspicious 
looking  colonies. 

Endo-Agar  must  be  kept  in  the  dark,  as  it  otherwise 
gradually  becomes  red.  The  Litmus  lactose  cigar,  pre- 
pared according  to  the  methods  of  Conradi  and  Drigalski, 
cannot  be  kept  for  any  length  of  time,  as  in  old  culture 
media  the  difference  between  typhoid-colonies  and  coli- 
colonies  does  not  appear  distinct  enough.  To  overcome 
this  disadvantage  the  culture  media  have  to  be  kept  with- 
out litmus-solution.  Crystal  violet  and  sugar  of  milk  and 
the  substances  are  added  only  shortly  before  use  (cf. 
Chapter  XII). 

Malachite  Green  Agar. — This  culture  medium,  accord- 
ing to  Loeffler  (cf.  QhapterXII),  contains  malachite-green 
so  concentrated  that  it  stops  the  growth  of  coli  bacteria  al- 
most entirely,  but  hardly  influences  the  growth  of  typhoid 


108     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

bacilli.     Thus  in  the  faeces  coli  bacilli  do  not  grow  at  all, 
or  very  scarcely  in  this  culture  medium. 

After  twenty- four  hours  the  typhoid-colonies  appear  in 
transmitted  light  delicate  and  transparent  and  are  macro-, 
scop ically  hardly  visible  (about  the  size  of  a  sand  grain), 
the  coli-colonies  are  thicker,  non-transparent,  of  a  whitish- 
cloudy  appearance. 

For  cultures,  taken  from  the  faeces,  Loeffler  recommends 
to  add  3  per  cent,  of  sterile  ox-gall  to  the  malachite-green. 
The  added  malachite-green  then  must  be  1.9  per  cent,  of 
a  0.2  per  cent,  pure  solution  of  malachite-green-crystals. 

Biological  Characteristics  of  Typhoid  Bacilli  Important  in 
Differential  Diagnosis. — Typhoid  bacilli  do  not  coagulate 
milk;  coli  bacilli,  however,  do  as  a  rule,  after  twenty- four 
to  forty-eight  hours. 

B.  fcecalis  alkaligenes,  dysentery  bacilli,  and  para- 
typhoid bacilli  also  do  not  coagulate  milk. 

The  growth  of  typhoid  bacilli  in  litmus-whey  (cf. 
Chapter  XII)  produces,  after  twenty-four  hours,  a  small 
amount  of  acid,  under  3  per  cent. ,  while  the  growth  of  coli 
bacteria  produces  more  than  7  per  cent,  decinormal  acid. 
Typhoid  tubes  show,  therefore,  only  a  slight  reddish  tinge, 
while  coli  tubes  are  bright  red.  Litmus-whey,  inoculated 
with  typhoid  bacilli,  remains  perfectly  clear,  while  that 
inoculated  with  coli  bacilli  becomes  evenly  clouded.  B. 
fcecalis  alkali  genes,  by  its  format  ion  of  alkali,  turns  litmus- 
whey  blue.  Dysentery  bacilli  and  type  A  of  the  para- 
typhoid bacilli  act  like  typhoid  bacilli,  while  type  B  pro- 
duces at  first  a  small  amount  of  acid,  but  after  a  few  days' 
growth  alkali. 

Growth  in  Barsiekoiv's  Culture  Medium  (cf.  Chapter 
XII). — In  BarsieTcoitfs  nutrose- sodium  chloride  solution, 
containing  1  per  cent,  grape-sugar,  typhoid  bacilli  and  coli 
bacilli  produce  considerable  acid  and  cause  coagulation, 


FAECES  109 

while  dysentery  bacilli,  at  least  during  the  first  few  days, 
produce  very  little  acid  and  do  not  cause  coagulation.  This 
solution  can,  therefore,  be  used  to  distinguish  between 
typhoid  and  dysentery  bacilli.  If,  instead  of  grape-sugar, 
1  per  cent,  milk-sugar  is  used,  typhoid  and  'coli  bacilli 
may  be  differentiated  by  means  of  this  culture  medium, 
since  typhoid  and  dysentery  bacilli  act  in  the  same  man- 
ner— i.e.,  they  both  leave  the  solution  unchanged,  while 
coli  bacilli  produce  acid  and  cause  coagulation.  If 
Barsiekow's  solution  contains  1  per  cent,  grape-sugar  and 
1  per  cent,  milk-sugar,  after  twenty-four  hours'  growth- 
Dysentery  tubes  show  acid  formation,  but  no  coagulation. 
Typhoid  tubes  show  acid  formation  and  clouding  due  to 

slight  coagulation. 
Coli  tubes  show  acid  formation  and  complete  coagulation. 

If  the  medium  containing  1  per  cent,  grape-sugar  is 
poured  into  fermentation-flasks,  at  the  end  of  thirty-six 
hours  the  following  conditions  are  present: 

Dysentery  tubes  show  acid  formation. 
Typhoid  tubes  show  acid  formation  and  coagulation. 
Coli  tubes  show  acid  formation,  coagulation,  and  gas  for- 
mation. 

Behavior  in  Culture  Media  Containing  Grape-Sugar.— 
Typhoid  bacilli,  dysentery  bacilli,  and  B.  fcecalis  alTcali- 
genes  do  not  ferment  grape-sugar,  while  most  varieties  of 
coli,  and  both  types  of  paratyphoid  bacilli  do  ferment  it 
with  the  formation  of  gas  (CO2). 

The  test  is  made  by  stab-culture  in  2  per  cent,  grape- 
sugar-agar,  or  by  the  inoculation  of  fermentation  flasks 
containing  2  per  cent,  grape-sugar-bouillon. 

Growth  in  Rothberger' s  Neutral  Red  Agar  (cf.  Chapter 
XII) :  Typhoid,  dysentery  bacilli  and  the  bacillus  fecalis 


110    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

BIOLOGICAL   CHARACTERISTICS   OF  TYPHOID 

BE    CONSIDERED   IN 


Behavior  in  — 

Bacteria 

Motilitv 

Litmus- 

Sugar- 

Milk 

Whey 

Agar 

Typhoid 
bacilli 

Motile 

No  coagu- 
lation 

Slight  acid 
rormation  ; 

No  fer- 
mentation 

clear 

Bacterium 

Non- 

Coagula- 

Liberal 

Fermenta- 

coli 

motile  or 

tion 

acid 

tion 

slightly 
motile 

formation  ; 
clouded 

Alkali- 
genes 

Motile 

No  coagu- 
lation 

Alkali 
formation 

No  fer- 
mentation 

Dysentery 
bacilli 

Non- 
motile 

No  coagu- 
lation 

Slight  acid 
formation  ; 

No  fer- 
mentation 

clear 

Para- 
typhoid 
bacillus  A 

Motile 

No  coagu- 
lation 

Slight  acic 
formation  ; 
clear 

Fermenta- 
tion 

Para- 

Motile 

No  coagu- 

At first, 

Fermenta- 

typhoid 
bacillus  B 

lation 

acid 
formation 

tion 

later, 

alkali 

formation 

F^CES 


111 


BACILLI   AND   OTHER   BACTERIA  WHICH   MUST 
DIFFERENTIAL  DIAGNOSIS 


Behavior  in  — 

Barsiekow's  Culture  Medium 

' 

Tnrlr»l 

Neutral-red- 
Agar 

1  Per  Cent. 

1  Per  Cent. 

1  Per  Cent. 
Grape- 

J.IKJ.O1 

Formation 

sutjar  • 

Grape-  Sugar 

Milk-Sugar 

i  nfcin  , 
1  Per  Cent. 

Milk-sugar 

No  reduc- 

Acid 

No  acid 

Acid 

No  indol 

tion;  no 

formation  ; 

formation  ; 

formation  ; 

formation 

fermenta- 
tion 

coagula- 
tion 

no  coagu- 
lation 

clouded 

Reduction  ; 

Acid 

Acid 

Acid 

Indol 

fermenta- 

formation ; 

formation  ; 

formation  ; 

formation 

tion 

coagula- 

coagula- 

coagula- 

tion 

tion 

tion 

No  reduc- 

No indol 

tion;  no 

formation 

fermenta- 

tion 

No  reduc- 

Slight acid 

Slight  acid 

Acid 

No  indol 

tion;  no 

formation  ; 

formation  ; 

formation  ; 

formation 

fermenta- 

no coagu- 

no coagu- 

clear 

tion 

lation 

lation 

Reduction  ; 

No  indol 

fermenta- 

formation 

tion 

Reduction  ; 

No  indol 

fermenta- 

formation 

tion 

112    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

alkaligenes  grow  in  this  culture  medium  without  changing 
it.  Bacteria  coli  and  paratyphoid  bacilli  discolor  it  after 
twenty- four  hours'  growth  by  reduction  of  the  pigment 
and  bring  about  a  greenish  fluorescence  and  gas  formation 
owing  to*  the  sugar  in  the  culture  medium.  The  test  is 
made  by  means  of  stab-cultures  in  well-filled  test-tubes 
or  still  better  with  shake-cultures. 

Growth  in  Loeffler^s  Green  Solution  (cf.  Chapter  XII). 
— After  having  grown  from  sixteen  to  twenty  hours  in  green 
Solution  Ij  the  typhoid  bacilli  produce  coagulation.  Next 
to  and  above  the  smooth  coagulum  is  a  clear  green  fluid. 
Coli  and  paratyphoid  bacilli  also  precipitate  the  nutrose, 
but  with  lively  gas  formation  owing  to  the  simultaneous 
fermentation  of  milk  and  grape-sugar;  it  does  not  form  a 
smooth  coagulum,  but  it  looks  torn  and  adheres  to  the  wall 
of  the  test-tubes  like  a  dirty  green  coating.  On  the  sur- 
face a  green  foam-ring  appears. 

Green  Solution  //is  not  changed  by  the  typhoid  bacilli, 
but  the  coli  bacteria  ferment  it,  and  the  changes  are  the 
same  as  in  green  Solution  I.  The  paratyphoid  bacilli  grad- 
ually discolor  themselves  from  light-green  into  a  pale- 
yellow  without  coagulating.  With  the  aid  of  these  two 
solutions,  the  coli  bacteria,  the  typhoid  and  paratyphoid 
bacilli  are  differentiated  from  each  other. 

Indol  Reaction. — Typhoid  bacilli,  in  contradistinction 
to  most  varieties  of  coli  bacilli,  produce  no  indol,  either 
when  grown  in  bouillon  or  peptone  water.  B.  fcecalis 
alkaligenes,  dysentery,  and  paratyphoid  bacilli  also  pro- 
duce no  indol. 

Detection  of  Indol. — To  10  cc  of  a  forty-eight-hour 
bouillon  or  peptone-water  culture  1  cc  of  a  0.02  per  cent, 
potassium  nitrite  solution  and  a  few  drops  of  chemically 
pure  concentrated  sulphuric  acid  are  added.  When  indol 
is  present  a  red  coloration  appears.  On  shaking  with 


FAECES  113 

amyi  alcohol  the  coloring  matter  is  extracted,  and  can  be 
more  clearly  seen.  It  is  well  always,  as  a  control,  to  place 
in  the  incubator  tubes  which  are  inoculated  with  an  authen- 
tic typhoid  culture  as  well  as  uninoculated  tubes. 

Order  of  Examination  of  the  Faeces  for  Typhoid 
Bacilli 

I.  Planting  of  Cultures  from  the  Faeces.— Thin  stools  of 
pasty  or  fluid  consistency  are  used  directly  for  planting 
cultures,  while  formed  stools  are  first  thoroughly  mixed 
with  a  small  quantity  of  sterile  physiological  salt  solution. 

1.  Onagarandthe  Conradi-Drigalski  culture  medium 
surface  colonies  are  planted;   for  this  purpose  a  right- 
angled  glass  spatula,  which  can  be  disinfected  by  burning 
with  alcohol,   or  the  ordinary  platinum  wire,    is  used. 
This    is   dipped  into  the  material  to  be  examined,  and 
rubbed  over  the  surface  of  the  plate  in  all  directions,  and 
then  smeared  in  the  same  manner  upon  a  second,  third, 
and  fourth  plate  without  being  again  sterilized  or  applied 
to  the  faeces.     In  this  manner  isolated  surface  colonies  are 
obtained  upon  plates  three  and  four.    The  Conradi-Drigal- 
i plates  should  remain  open  for  some  time  after  the  inocu- 
lation, until  they  have  become  absolutely  dry,  in  order  to 
guard  against  the  coalescence  of  the  developing  colonies. 
The  plates  are  then  placed  upside  down — that  is,  with  the 
cover  down — in  an  incubator  at  87°  C. 

2.  Inoculation  of  Urine- Gelatine. — After  this  has  been 
liquefied  "  dilution  plates"  are  made  in  the  usual  manner 
—i.e.,  the  first  tube  is  inoculated  with  two  loops  of  faeces. 
With  four  loops  from  this  a  second  tube  is  inoculated,  and 
with  six  to  eight  loops  from  the  second  tube  a  third  is  in- 
oculated.     The  inoculated  gelatine  is  then  poured  into 
plates,  which  after  the  medium  has  solidified  on  ice,  are 
placed  in  an  incubator  at  21.5°  to  22°  C. 


114     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

It  is  always  well  to  inoculate  several  series  of  agar  and 
Conradi-Drigalslci  plates. 

•  II.  Examination  of  the  Plates—  On  the  day  after  the 
inoculation  the  plates  are  tested  in  the  following  manner : 

Agar  and  Conradi-Drigalslci  Plates. — From  the  agar 
plates  only  the  small,  transparent,  bluish  iridescent  colo- 
nies need  be  considered  for  further  examination;  from  the 
Conradi-Drigalslci  plates  only  the  smallr  blue,  sharply 
outlined  colonies  resembling  dew-drops.  Very  minute 
portions  are  removed  from  these  with  a  platinum  needle, 
and  examined  in  hanging-drops.  When  motile  rods, 
having  the  appearance  of  typhoid  bacilli,  are  seen,  the 
remainder  of  the  colony  is  transplanted  upon  slanting  agar 
to  obtain  a  pure  culture.  The  so-called  "  preliminary 
agglutination  test' '  may  also  be  made  at  this  time. 

It  is  always  necessary  to  remove  a  large  number  of  sus- 
picious looking  colonies  from  the  plates  and  to  culture 
them  for  pure  culture  media. 

Cultures  made  according  to  Lentz  and  Tietz  are  exam- 
ined as  follows :  The  blue  plates  are  examined  first  for 
typhoid  bacilli  after  they  have  been  in  the  incubator  sixteen 
to  twenty  hours.  If  no  suspicious  colonies  are  found,  about 
8  to  10  cc  of  0.85  per  cent.  NaCl-solution  are  poured  over 
the  green  plate  which  has  remained  in  the  incubator  for 
twenty-four  hours,  and  is  then  left  standing  quite  still  for 
two  minutes.  The  fluid  is  now  moved  about  on  the  plate 
a  few  times,  whereby  the  loose  typhoid  and  paratyphoid 
colonies  are  separated  and  left  floating  in  the  fluid,  while 
the  thick  clusters  of  coli-colonies  are  segregated  in  toto  and 
soon  sink  to  the  bottom.  In  order  to  produce  the  latter 
effect  the  plate  is  tilted,  so  that  the  fluid  runs  up  to  the 
edge;  the  thick  clusters  fall  to  the  bottom  after  about 
half  a  minute,  one  to  three  loops  are  removed  from  the 
supernatent  fluid  and  planted  upon  a  Conradi-Drigalski 


F^CES  115 

plate  (each  loop  is  2  mg)  and  rubbed  up  with  a  glass 
spatula  on  this  and  another  blue  plate  according  to  the 
thickness  of  the  colonies.  If  paratyphoid  is  suspected, 
malachite-green  plates  are  used,  in  order  to  easier  recog- 
nize the  colonies  of  paratyphoid  B.  These  plates  are  ex- 
amined, after  standing  in  the  incubator  sixteen  to  twenty 
hours. 

The  pure  cultures  thus  obtained  are  inoculated  the  day 
after  for  the  purpose  of  determining  their  biological  quali- 
ties in  Loeffler's  green  solutions,  litmus- whey,  neutral 
red-agar;  furthermore  the  quantitative  macroscopic  ag- 
glutination-test is  made  with  a  high-potency  animal-im- 
mune-serum (cf.  p.  272). 

We  recognize  as  typhoid  bacilli  such  bacteria  which 
correspond  with  these  in  their  biological  qualities  and  are 
agglutinated  from  a  diluted  solution  of  a  high  potency  im- 
mune-serum which  is  of  about  the  same  titre  as  the  serum. 

We  must,  however,  take  note  of  the  fact,  that  freshly 
inoculated  typhoid  bacilli  very  often  do  not  agglutinate 
at  all  or  with  great  difficulty,  and  that  only  after  several 
inoculations  on  agar  may  agglutinate  easier  (cf.  p.  272). 

Paratyphoid  bacilli  are  found  in  diseases  which  re- 
semble typhoid  or  acute  gastro-enteritis. 

We  distinguish  two  types,  paratyphoid  bacillus  A  and 
B,  of  which  the  latter  is  found  very  frequently,  but  the 
first  extremely  rarely. 

Both  types  of  bacteria  look  under  the  microscope  like 
typhoid  bacilli,  but  they  differ  from  them  and  from  each 
other  by  their  behavior  in  culture-media,  by  their  reac- 
tion to  immunization,  and  by  their  animal  pathogenesis. 

The  culture  qualities  of  the  paratyphoid  bacilli  have 
already  been  discussed  when  speaking  of  the  differential 
diagnosis  of  the  typhoid  bacilli  (cf.  also  table  on  pp. 
110  and  111). 


116     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

The  paratyphoid  bacilli  A  and  B  are  distinguished 
positively  from  each  other  and  from  the  other  groups  of 
typhoid  coli  by  the  agglutination-test  with  a  high  potency 
immune-serum. 

Typhoid  bacilli  quite  occasionally  are  influenced  up  to 
a  certain  degree  by  typhoid  sera  and  typhoid  bacilli  by 
paratyphoid  sera,  this  is,  however,  observed  only  in 
relatively  concentrated  solutions  of  sera  and  depends  upon 
the  presence  of  common  partial  agglutination. 

In  contradistinction  to  the  typhoid  bacilli  the  para- 
typhoid bacillus  B  is  highly  pathogenic  for  some  animals, 
especially  guinea-pigs  and  mice,  and  even  very  small  doses 
are  sufficient  to  deadly  infect  these  animals  ^J^r  to 


loop  for  an  intraperitoneal  injection,  ^  to  •£$  loop  for  a  sub- 
cutaneous inoculation.     The  animals  die  of  septicemia.  j 
The  paratyphoid  bacillus  A  kills  mice  when  mixed  with 
the  food. 

The  paratyphoid  bacilli  are  demonstrated  in  the  faeces  ; 
the  same  way  as  the  typhoid  bacilli. 

As  culture  media   are   used   likewise   litmus-lactose,  1 
fuchsine-agar  and  malachite-green  agar. 

The  paratyphoid  bacillus  A  grows  in  these  media  the 
same  way  as  the»typhoid  bacilli.     Paratyphoid  bacillus  B  ! 
forms  blue  colonies  on   Conradi-Drigalski  plates,   which  ! 
mostly,  but  not  as  a  rule,  are   larger,  juicier,   and  less  1 
transparent  than  typhoid-colonies. 

The  colonies  of  paratyphoid  bacillus  B  appear  colorless 
on  fuchsine-agar,  the  same  as  the  colonies  of  the  typhoid  j 
bacilli,  but  they  are  occasionally  larger  and  fuller  devel- 
oped. 

Paratyphoid  bacillus  B  forms  in  malachite-green  agar 
after  having  grown  in  the  incubator  sixteen  to  twenty 
hours,  transparent,  slightly  milky  colonies  of  2  to  3 
millimetres  diameter  which  stain  the  zone  around  them 


F^CES  117 

yellow.  The  malachite-green  agar  offers  especially  to  the 
paratyphoid  bacillus  B  very  favorable  conditions  to  grow, 
and  is,  therefore,  very  serviceable  for  cultures  from  the 
faeces,  especially  if  they  have  gone  through  the  prelim- 
inary culture  according  to  Lentz  and  Tietz. 

The  paratyphoid  bacilli  are  identified  by  their  cultural 
qualities,  by  their  pathogenity  in  animals,  and  by  the 
agglutination  test  with  a  high  potency  immune  serum. 

Bacilli  in  Meat-Poisoning   (Enteritis   Bacilli) 

The  bacteria  found  in  meat  poisonings  under  gastro- 
intestinal symptoms  may  be  divided  into  two  groups. 

The  first  group,  type  Aertryck  (according  to  De 
Nobili))  has  the  same  cultural  qualities  as  paratyphoid 
bacillus  B  and  cannot  be  separated  from  it  by  the  reac- 
tions for  immunization.  They  may  be  bacilli,  which  are 
identical  with  the  paratyphoid  bacillus  B.  The  bacilli  of 
the  so-called  hog-cholera  group  (B.  typhi  murium  Loeffler, 
bacillus  of  the  hog-pest,  psittacose-bacillus)  also  belong  to 
this  group. 

The  second  group,  type  Gaertner  (according  to  De 
NoMli),  is  also  culturally  identical  with  the  paratyphoid 
bacillus  B,  but  differs  from  it  by  its  sero-diagnosis.  In 
this  group  also  belong  the  rat-pathogenic  bacilli  of 
Danysz,  Issatsclienko,  and  Dun&ar's  rattin.  The  detec- 
tion of  these  bacteria  is  accomplished  by  the  method  given 
for  typhoid  and  paratyphoid. 

Dysentery  Bacilli 

Based  upon  numerous  investigations  carried  out  withih 
the  last  few  years,  it  can  be  assumed  that  in  all  proba- 
bility the  bacillus,  cultivated  first  by  Shiga,  and  two  years 
later  by  Kriise,  from  the  bloody  mucus  evacuations  of 
dysentery  patients,  is  the  exciting  cause  of  the  epidemic 


118    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

dysentery,  which  appears  chiefly  in  countries  of  the  North 
Temperate  Zone. 

This  disease  must  not  be  confused  with  that  caused  by 
amoebae,  now  usually  designated  as  amoebic  dysentery. 

Characteristics  of  Dysentery  Bacilli — Dysentery  bacilli 
are  rods  about  the  length  of  typhoid  bacilli,  but  somewhat 
thicker  and  plumper.  In  contradistinction  to  the  latter, 
they  are  non-motile.  They  stain  easily  with  dilute  aniline 
dyes,  and  are  decolorized  by  Gram. 

The  dysentery  bacilli  grow  in  all  the  usual  media. 
Their  cultures  develop  a  distinct  odor  after  sperma.  They 
form  the  same  as  the  typhoid  bacilli  in  agar  round,  flat 
cultures,  which  are  whitish  in  ordinary  light  and  moist, 
in  transmitted  light  bluishly  iridescent. 

If  grown  in  gelatine  their  superficial  colonies  after 
forty-eight  hours  are  similar  to  those  of  the  typhoid  bacilli. 
They  also  show  the  so-called  wine-leaf  shape.  They  do  not 
liquefy  gelatine.  If  grown  on  potatoes  which  are  of  a 
weak  acid  reaction,  or  in  bouillon,  they  do  not  differ  from 
typhoid  bacilli.  In  the  culture  media  of  Conradi-Drigalski 
they  form  round  dewdrop-like  colonies,  which  show  a  faint 
milky  cloudiness,  and  they  do  not  change  the  blue  color 
of  the  culture  media. 

As  to  the  biological  characteristics  of  the  dysentery 
bacilli  cf.  pp.  110  and  111. 

They  are  distinguished  from  the  typhoid  bacillus  be- 
cause of  their  immotility  and  their  behavior  in  Barsiekow^s 
solution;  from  the  paratyphoid  bacillus  because  of  their 
behavior  in  sugar  and  neutral  red  agar;  from  the  coli 
bacilli  because  of  their  growth  in  milk,  litmus-whey,  sug- 
ar agar,  neutral  red  agar,  etc.  (cf.  table,  pp.  110  and  111). 

For  the  positive  identification  of  the  dysentery  bacillus 
is  the  agglutination  with  a  specific  serum  of  a  high 
potency. 


FAECES  119 

The  SMga-Kruse  bacillus  is  to  be  distinguished  from 
the  Flexner  and  the  pseudo-dysentery  bacilli  through  its 
behavior  in  nutritive  media,  which  contain  mannit.  The 
SMga-Kruse  bacillus  does  not  decompose  mannit;  the 
Flexner  and  the  pseudo-dysentery  bacilli  cause  fermenta- 
tion of  the  mannit  with  the  production  of  acids  without 
the  production  of  gases.  To  test  this  behavior  we  use 
Barsiekovfs  nutritive  solution  with  one  per  cent,  mannit 
or  the  litmus  mannit  agar  (cf.  Chapter  XII).  The  SMga- 
Kruse  bacillus  does  not  change  the  color  of  the  Barsie- 
koiv  solution,  the  other  bacteria  color  the  solution  red 
and  coagulate  the  albumin.  In  litmus-mannit-agar  stab- 
cultures  may  be  made.  The  SMga-Kruse  bacillus  reduces 
the  litmus  to  a  lighter  color  in  the  deeper  layers ;  the  Flex- 
ner and  the  pseudo-dysentery  bacillus  color  the  solution  a 
reddish  violet  after  twenty-four  hours,  and  a  distinct  red 
after  forty-eight  hours.  Instead  of  stab-cultures  we  can 
also  get  the  cultures  upon  the  surface  of  this  nutritive 
medium  which  has  been  put  in  Petri  dishes.  The  colo- 
nies of  the  SMga-Kruse  bacilli  leave  the  color  of  the 
nutritive  media  unchanged,  the  Flexner  and  the  pseudo- 
dysentery  bacilli  stain  the  media  red  after  forty-eight 
hours. 

Another  means  of  differentiation  is  the  agglutination 
test.  In  a  serum  produced  by  the  SMga-Kruse  bacilli, 
the  Flexner  bacilli  are  agglutinated  only  in  concentrated 
solutions,  and  vice  versa.  It  is  therefore  necessary  to  have 
a  thorough  titration  of  the  serum  in  order  to  be  able  to 
make  a  positive  diagnosis. 

In  the  animal  experiments  the  Shiga-Kruse  bacilli 
proved  more  poisonous  than  the  Flexner  and  pseudo- 
dysentery  bacilli.  The  intravenous  injection  of  one- 
twentieth  per  cent,  of  the  living  culture,  or  of  a  60 
per  cent,  of  the  dead  culture  of  the  SMga-Kruse  bacilli 


120    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

kill  rabbits  in  from  one  to  three  days,  producing  par- 
alysis. 

The  dysentery  bacilli  are  easily  demonstrated  so  long 
as  the  faeces  contain  blood  and  mucus.  Occasionally  the 
examinations  must  be  repeated  several  times.  If  the  evac- 
uations have  again  become  of  fecal  consistency  they  are 
not  so  easily  or,  possibly  not  at  all,  demonstrable.  The 
best  nutritive  medium  is  the  Conradi-Driyalski  agar,  in 
which  the  sugar  of  milk  was  substituted  by  mannit  of  a 
like  concentration. 

Order  of  Examination. — A  smear  is  made  from  a  mucus 
fleck  and  stained  with  dilute  carbol-fuchsin.  Dysentery 
bacilli  are  frequently  detected  in  it  in  almost  pure  culture, 
only  a  few  coli  bacilli,  as  a  rule,  being  present  with  them. 
Other  mucus  flecks  are  used  for  the  inoculation  of  cultures. 
Gelatine  plates  are  made  in  the  usual  manner,  and  smears 
upon  agar  and  the  Conradi-Drigalski  culture  medium,  as 
in  the  examination  of  the  faeces  for  typhoid  bacilli. 

The  gelatine  plates  are  examined  forty-eight  hours 
after  inoculation,  the  agar  plates  sixteen  to  twenty-four 
hours.  Pure  cultures  are  obtained  from  the  suspicious- 
looking  colonies,  and  are  tested  for  their  biological  charac- 
teristics, and  the  agglutination  test  is  carried  out  with  a 
high-potency  immune  serum  (cf.  Examination  of  Typhoid 
Plates,  p.  114). 

Cholera  (Plate  V,  Fig.  H) 

Characteristics  of  the  Cholera  Vibriones. — Cholera  vibri- 
ones  are  very  motile,  slightly  curved,  short  rods,  which 
stain  easily  with  dilute  aniline  dyes  (carbol-fuchsin,  1  to 
10),  and  are  decolorized  by  Gram.  In  stained  smears 
from  pure  cultures  numerous  bacilli  lying  close  together 
in  semicircular  or  S-shaped  figures,  or,  especially  in  old 
cultures,  in  spirally  interwoven  threads,  are  frequently 


FAECES  121 

seen.  Cholera  vibriones  grow  easily  upon  all  the  usual 
culture  media,  especially  in  the  presence  of  a  marked 
alkaline  reaction. 

Upon  agar  they  develop,  after  eighteen  to  twenty-four 
hours'  growth  at  87°  C.,  small  transparent  colonies,  which, 
when  held  against  the  light,  have  a  bluish  iridescence. 
They  can  be  easily  distinguished  from  colonies  of  most  of 
the  other  bacteria  present  in  cultures  from  the  faeces  by 
their  transparency  when  examined  in  direct  light. 

On  gelatine,  after  twenty-four  hours'  growth  at  22°  C., 
cholera  colonies  appear  to  the  naked  eye  as  very  small, 
bright  points.  When  examined  with  the  low  power,  they 
appear  as  small,  round,  glistening  discs  with  irregular 
wavy  margins.  The  surface  of  the  colonies  is  granular 
and  highly  refractive,  so  that  they  appear  as  though 
sprinkled  with  fine  particles  of  glass.  Gelatine  is  slowly 
liquefied. 

In  bouillon,  cholera  vibriones  grow  luxuriantly,  cloud- 
ing it  evenly,  and  forming  a  film  on  its  surface. 

Milk  is  not  coagulated;  blood-serum  is  liquefied. 

Alkaline  peptone-water  is  an  especially  favorable  cul- 
ture medium  for  cholera  vibriones,  as,  in  fact,  for  all 
vibriones.  When  material  which  contains  other  micro- 
organisms in  addition  to  cholera  bacteria  is  placed  in 
peptone-water,  a  marked  increase  of  the  vibriones  takes 
place,  especially  in  the  upper  portion  of  the  solution,  where 
they  grow  much  faster  and  more  luxuriantly  than  the  other 
bacteria  present.  Frequently,  even  after  but  six  hours' 
growth  at  37°  C.,  very  motile  aerobic  vibriones  are  present 
in  pure  culture  on  the  surface  of  the  culture  media. 

If  a  few  drops  of  concentrated,  chemically  pure  sul- 
phuric acid  are  added  to  a  twenty- four  hours'  peptone- 
water  culture  of  cholera  vibriones,  a  violet  coloration  ap- 
pears^ which  may  be  extracted  by  shaking  with  amyl 


122    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

alcohol  (cholera-red  reaction).  This  color  reaction 
depends  upon  the  fact  that  the  bacteria  when  grown  in 
peptone- water  produce  a  large  amount  of  indol,  and  reduce 
the  nitrates  present  in  the  media  to  nitrites.  Upon  the 
addition  of  sulphuric  acid,  nitrous  acid  is  formed,  which 
unites  with  the  indol  and  produces  the  red  color  (nitroso- 
indol  reaction).  This  reaction  is  by  no  means,  as  was 
formerly  thought,  a  peculiarity  of  cholera  vibriones,  but  is 
produced  by  a  number  of  other  vibriones.  It  has,  how- 
ever, a  value  in  differential  diagnosis,  as  it  is  a  constant 
characteristic  of  cholera  vibriones,  and  its  absence,  there- 
fore, is  proof  that  the  bacteria  examined  are  not  cholera 
vibriones.  Of  course,  a  control  must  always  be  made  to 
see  if  an  authentic  cholera  culture  gives  the  red  reaction 
when  grown  in  a  tube  of  the  same  peptone-water. 

Serum  diagnosis  affords  the  most  valuable  means  for 
differentiating  between  cholera  vibriones  and  other  closely 
related  varieties  of  vibriones  which  may  be  present  in  the 
faeces  with  them.  Only  such  vibriones  as  are  agglutinated 
by  a  high  dilution  of  a  serum  obtained  from  an  animal 
immunized  with  cholera  vibriones,  and  are  dissolved  by 
the  bacteriolytic  elements  of  such  a  serum,  in  the  manner 
prescribed  in  Pfeiffer^s  test,  should  be  considered  as  true 
cholera  vibriones.1 

The  agglutination  test  is  carried  out  in  the  manner 
described  under  Examination  of  the  Faeces  for  Typhoid 
Bacilli. 

Pfeiffer's  test  depends  upon  the  fact  that  when  an  ani- 
mal is  immunized  with  cholera  vibriones,  in  addition  to 
agglutinin,  bacteriolytic  substances  appear  in  the  serum. 
If  such  immune  serum  is  injected  together  with  cholera 

1  Serum  used  for  the  agglutination  test  and  for  Pfeiffer's  test  can 
be  obtained  from  the  Institut  fur  Infektionskrankheiten  in  Berlin. 


FvECES  123 

vibriones  into  the  peritoneal  cavity  ol  a  guinea-pig,  and 
after  twenty  minutes  to  one  hour  a  few  drops  of  the  peri- 
toneal contents  are  withdrawn  with  a  capillary-tube  and 
examined  in  a  hanging-drop,  vibriones  are  no  longer 
found,  but  in  their  place  small,  pale  spherules.  Finally, 
these  disappear  and  the  cholera  vibriones  are  completely 
dissolved  by  the  bacteriolytic  substances  contained  in  the 
serum.  This  reaction  is  absolutely  reliable,  as  the  bac- 
teriolytic action  of  cholera  immune  serum  is  directed  only 
against  cholera  vibriones,  never  against  other  bacteria. 

Method  of  Carrying  Out  Pfeiffer's  Test 

The  serum  used  for  this  test  should  have  as  high  a 
potency  as  possible;  this  should  be  at  least  so  high  that 
0.0002  gramme  is  sufficient  to  dissolve,  within  half  an 
hour  with  the  formation  of  spherules,  the  cholera  bacteria 
contained  in  a  mixture  of  one  loop  (1  loop  =  2  milli- 
grammes) of  an  eighteen  hours'  agar  cholera  culture  of 
standard  virulence,  with  1  cc  of  nutrient  bouillon,  when 
injected  into  the  peritoneal  cavity  of  a  guinea-pig — i.e., 
the  serum  must  have  a  titre  (standard  of  potency)  of  at 
least  0.0002  gramme. 

For  Pfeiffer^s  test,  four  guinea-pigs,  weighing  200 
grammes  each,  are  necessary. 

Animal  A  receives  five  times  the  titre  dose — i.e.,  1 
milligramme  of  a  serum  whose  titre  is  0.0002  gramme. 

Animal  B  receives  ten  times  the  titre  dose — i.e.,  2 
milligrammes  of  the  serum. 

Animal  C  serves  as  control-animal  and  receives  fifty 
times  the  titre  dose — i.e.,  10  milligrammes  of  normal 
serum  from  the  same  kind  of  animal  as  that  from  which 
the  serum  used  with  animal  A  and  B  was  obtained. 

Each  animal  receives  the  serum  mixed  with  one  loop  of 
the  culture  to  be  examined,  grown  on  agar  for  eighteen 


124    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

hours  at  87°  C.,  in  1  cc  of  bouillon  (not  in  salt  solution 
or  peptone  solution),  injected  into  the  peritoneal  cavity. 

Animal  D  receives  a  quarter  of  a  loop  of  the  cholera 
culture  intraperitoneally,  in  order  to  ascertain  if  the  cul- 
ture is  virulent  for  guinea-pigs.  A  blunt  cannula  is  used 
for  the  injection.  The  injection  is  made  into  the  peri- 
toneal cavity  through  a  cut  in  the  skin ;  the  cannula  can 
be  easily  forced  into  the  peritoneal  cavity.  The  peritoneal 
exudate  is  withdrawn  for  microscopical  examination,  at 
the  same  point,  by  means  of  a  capillary  glass-tube. 

The  exudate  is  examined  in  a  hanging-drop,  with  the 
high  power,  twenty  minutes  after,  and  one  hour  after,  the 
injection. 

In  animal  A  and  animal  B,  after  twenty  minutes  or, 
at  the  latest,  after  one  hour,  the  typical  spherules  must 
have  formed  or  the  vibriones  must  have  dissolved ;  while 
in  animals  C  and  D  a  great  quantity  of  highly  motile, 
well-preserved  vibriones  must  be  present.  For  the  identi- 
fication of  convalescent  cases  of  cholera,  Pfeiffer's  reac- 
tion must  be  carried  out  in  the  following  manner:  Dilu- 
tions of  serum  from  the  suspected  person,  in  proportions 
of  1 :  20,  1 : 100,  1 :  500,  are  made  with  bouillon.  From 
these  1  cc  is  taken,  mixed  with  one  loop  of  an  eighteen- 
hour  agar  culture  of  virulent  cholera  vibriones,  and  injected 
into  the  peritoneal  cavity  of  guinea-pigs  weighing  200 
grammes  each.  A  control-animal  receives  an  intraperi- 
toneal  injection  of  a  quarter  of  a  loop  of  the  same  culture, 
dissolved  in  1  cc  of  bouillon,  but  without  serum.  A  posi- 
tive result  ofj  the  reaction  after  twenty  to  sixty  minutes 
indicates  that  the  person  from  whom  the  serum  was  taken 
has  had  cholera.  (Instructions  of  the  Prussian  Ministry 
entrusted  with  the  Control  of  Religious^  Educational,  and 
Medical  Affairs,  in  Regard  to  the  Bacteriological  Diagnosis 
of  Cholera,  November  6,  1902. ) 


FAECES  125 

Detection  of  Cholera  Vibriones  in  the  Faeces 

Detailed  instructions  concerning  the  examination  of 
the  fseces  for  cholera  bacteria  are  contained  in  the  above- 
mentioned  order. 

1.  Microscopical  Examination. — Smears  are  made,  when 
possible,  from  a  mucus  fleck,  and  stained  with  dilute  car- 
bol  fuchsin  (1:9).     Frequently,  typical  comma  bacilli  are 
present  in  these  smears  in  great  numbers,  or  even  in  pure 
culture,  arranged  in  characteristic  shoals.     In  many  cases, 
however,  the  bacteria  are  not  present  in  such  large  num- 
bers, and  cannot  be  recognized  among  the  great  number 
of  bacteria  normally  present  in  the  intestines. 

In  addition,  hanging-drops  are  made  from  a  sputum 
fleck  with  peptone  solution,  and  examined  fresh  and 
stained,  at  once  and  after  half  an  hour,  in  an  incubator 
at  87°  C.  Occasionally  the  vibriones  are  seen  to  collect  at 
the  margin  of  the  drop. 

A  certain  diagnosis  can  never  be  made  from  the  micro- 
scopical examination  alone,  but  cultural  procedures  must 
always  be  used. 

2.  Gelatine  Plates. — Two  tubes  of  melted  gelatine  are 
inoculated  with  one  loop  of  the  material  to  be  examined 
(when  possible,  from  a  mucus  fleck)  and  two  dilutions 
made  in  the  usual  manner  by  transplanting  three  loops  at 
a  time.     The  gelatine  is  poured  into  plates  and  examined 
after  eighteen  hours'  growth  at  22°  C. 

3.  Agar  Plates. — Agar  plates  must  be  absolutely  dry. 
They  are,   therefore,  placed,  open  and  with  the  surface 
down,  in  an  incubator  for  half  an  hour  before  they  are  in- 
oculated.     A  loop  from  the  faeces  or  a    mucus  fleck    is 
planted  upon  a  series  of  plates,  in  the  manner  described 
under  the  examination  for  typhoid  bacilli.     The  plates  are 
examined  after  twelve  to  eighteen  hours'  growth  at  37°  C. 


126    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

4.  Enrichment  by  Means  of  Peptone  Solution. — Six  tubes, 
containing  10  cc  each,  are  inoculated;  each  tube  receiving 
one  loop  of  faeces.  When  it  is  suspected  that  very  few 
cholera  vibriones  are  present  in  the  stools,  1  cc  of  faeces  is 
covered  in  a  flask  with  50  cc  of  peptone  solution.  After 
six  to  twelve  hours'  growth  in  an  incubator  at  87°  C.,  a 
small  portion  is  withdrawn  from  the  surface  of  the  peptone 
solution  without  disturbing  the  rest,  and  examined  micro- 
scopically. Frequently,  a  pure  culture  of  vibriones  is  seen 
in  the  specimen.  Gelatine  and  agar  plates  are  inoculated 
from  the  tube  containing  the  most  suspicious-looking  bac- 
teria. If  pure  cultures  do  not  grow  on  these  plates,  the 
suspicious-looking  colonies  are  removed  and  transplanted 
on  agar  tubes. 

Finally,  serum  tests  are  carried  out  with  the  pure  cul- 
tures (quantitative  macroscopical  agglutination  test  and 
Pfeiffer '  s  reaction  ) .  It  must  be  remembered  that  vibriones 
resembling  cholera  vibriones,  which  may  be  present  in  the 
faeces,  are  also  increased  in  peptone-water,  and  that  they 
cannot  be  distinguished  upon  agar  and  gelatine  from  true 
cholera  vibriones. 

The  diagnosis  of  cholera  is  considered  certain  when  all 
these  tests  are  positive. 

The  first  cases  of  an  epidemic  should  always  be 
examined  in  this  thorough  manner.  Later  in  the  epi- 
demic, cultural  examination  and  the  preliminary  ag- 
glutination test  in  a  hanging-drop  (cf.  p.  272)  suffice, 
when  the  latter  gives  a  definite  result. 


Tubercle  Bacilli 

The  detection  of  tubercle  bacilli  in  the  faeces  is  accom- 
plished by  means  of  stained  smears.  It  succeeds  most 
easily  in  smears  made  from  the  flecks  of  mucus  and  pus, 


FAECES  127 

which  are  present  in  the  diarrhoeal  evacuations  of  patients 
suffering  from  intestinal  tuberculosis. 

If  the  faeces  to  be  examined  are  formed,  they  are 
(according  to  Strassburger)  mixed  with  water  and  cen- 
trifugalized.  The  cloudy  liquid  above  the  sediment 
is  poured  off  and  diluted  with  96  per  cent,  alcohol  (two 
parts  fluid  to  be  examined,  one  part  alcohol).  It  is 
then  centrifugalized  again  and  smears  made  from  the 
sediment. 

Care  should  be  exercised  in  forming  an  opinion  from 
the  smears.  Aside  from  the  fact  that  a  negative  result 
is,  of  course,  in  no  case  proof  against  tuberculosis,  it 
should  always  be  borne  in  mind  when  the  result  is  posi- 
tive that  the  tubercle  bacilli  may  have  gained  entrance  to 
the  intestines  in  sputum  which  has  been  swallowed. 
Moreover,  acid- fast  bacilli  which  were  not  tubercle  bacilli, 
have  been  repeatedly  found  in  the  faeces. 

The  case  is  in  all  probability  one  of  intestinal  tu- 
berculosis, when  in  repeated  examinations  numerous  acid- 
fast  bacilli,  having  the  appearance  of  tubercle  bacilli,  are 
found  in  the  fasces  of  patients  who  have  certainly  swal- 
lowed no  sputum. 


Staphylococci  and  Streptococci 

These  bacteria  appear  in  the  faeces  both  as  the  ex- 
citing cause  of  acute  intestinal  catarrh,  and  following 
the  rupture  of  abscesses  into  the  intestines.  In  the 
first  case,  the  pyogenic  bacteria  are  seen  in  such  great 
numbers  that  the  micro-organisms  normally  present 
<in  the  stools  are  completely  overshadowed  by  them. 
Their  detection  is  accomplished  by  means  of  smears 
stained  with  dilute  carbol-fuchsin,  and  according  to 
Gram. 


128    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Anthrax  Bacilli 

In  the  rare  cases  of  intestinal  anthrax,  anthrax  bacilli 
are  evacuated  in  the  faeces.  Their  detection  is  accom- 
plished by  means  of  cultures.  Cultures  are  planted  upon 
gelatine  and  agar,  the  characteristic  colonies  (cf.  p.  300) 
are  removed,  grown  in  pure  culture,  and  for  their  identifi- 
cation injected  into  white  mice. 

Plague  Bacilli 

Plague  bacilli  have  been  found  in  a  few  cases  in  the 
faeces  of  plague  patients.  Their  detection  is  accomplished 
by  means  of  animal  inoculation,  the  faeces  being  rubbed 
into  the  shaved  abdomen  of  guinea-pigs 


CHAPTER  VII 
EXAMINATION  OF  THE  URINE 

I.  Collection  of  the  Urine 

The  twenty- four  hours'  quantity  is  usually  collected  in 
a  receptacle  which  has  been  thoroughly  cleansed  with  hot 
water.  To  guard  against  the  decomposition  of  the  urine, 
it  is  well  to  add  a  piece  of  thymol  the  size  of  a  bean,  or 
10  to  20  drops  of  chloroform.  If,  in  spite  of  this,  the 
urine  decomposes  quickly,  and  shows  a  pronounced  alka- 
line reaction,  it  is  well  to  request  a  freshly  passed  sample 
of  urine  for  examination,  in  addition  to  the  twenty-four 
hours'  sample.  In  many  cases  it  is  necessary  to  examine 
different  portions  of  the  daily  output  separately.  In  affec- 
tions of  the  kidney  the  morning  and  evening  urine  are 
separately  examined;  in  mild  cases  of  diabetes  or  in  ali- 
mentary glycosuria,  that  passed  before  and  after  meals. 
When  there  is  suspicion  of  a  physiological  or  cyclic  albumi- 
nuria,  every  sample  of  urine  passed  must  be  separately 
examined  for  albumin.  For  purposes  of  differential  diag- 
nosis in  diseases  of  the  urethra  and  bladder,  a  single 
sample  (preferably  the  first  morning  urine)  is  collected  in 
two  or  three  vessels,  and  each  portion  examined  separately 
for  its  physical,  chemical,  and  microscopical  characteris- 
tics. Catheterization  of  the  ureters,  which  has  been 
recently  introduced  into  practice,  renders  it  possible  to 
isolate  the  urine  of  each  kidney,  and  by  this  means  to  de- 
termine with  certainty,  in  cases  of  unilateral  disease, 
which  of  the  two  kidneys  is  affected.  For  this  purpose 

129 


130    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

the  so-called  segregators,  by  means  of  which  the  bladder 
is  divided  into  two  parts  by  a  partition,  are  also  used. 
This  latter  method  is,  however,  not  entirely  free  from  ob- 
jections, and  in  cases  in  which  catheterization  of  the 
ureters  is  not  contraindicated,  it  is  to  be  preferred  to  the 
use  of  the  segregator.  In  collecting  urine  care  must 
always  be  taken  that  no  foreign  matter  (sputum,  menstrual 
blood,  spermatic  fluid,  etc. )  becomes  accidentally  mixed 
with  it. 

Concerning  the  Collection  of  Urine  for  Bacteriological 
Examination,  cf.  p.  237. 

II.  The  Identification  of  a  Fluid  as  Urine 

In  practice,  it  is  occasionally  necessary  to  be  able  to 
identify  with  certainty  as  urine  a  fluid  which  has  been 
submitted  for  examination.  This  may  be  necessary  in 
many  cases,  owing  to  the  suspicion  of  accidental  or  inten- 
tional substitution  on  the  part  of  the  patient.  In  other 
cases  the  identification  of  a  fluid  as  urine  is  absolutely 
indispensable  for  purposes  of  differential  diagnosis.  This 
latter  is  especially  true  of  fluids  aspirated  from  the  region 
of  the  kidneys.  In  such  cases  the  differential  diagnosis 
lies  usually  between  hydronephrosis  and  cystic  tumors,  or 
echinococcus. 

To  identify  a  fluid  as  urine,  some  of  the  constituents 
characteristic  of  urine  alone  must  be  detected  in  it.  Of 
the  numerous  organic  and  inorganic  constituents  of  the 
urine,  urea  and  uric  acid  are  considered  characteristic, 
and  their  simultaneous  presence  in  a  fluid  is  recognized 
as  sufficient  evidence  that  the  fluid  is  urine.  If,  in  addi- 
tion, a  third  constituent  of  the  urine — creatinin — is  de- 
tected, the  fluid  examined  may  be  considered  with  cer- 
tainty as  urine.  These  constituents  of  the  urine  are 
detected  in  the  following  manner : 


URINE  131 

Urea:  CO(NH2)2. — Twenty-five  to  50  cc  of  urine  are 
evaporated  in  a  porcelain  dish  to  a  thin  syrup.  After  this 
has  cooled,  a  few  cc  of  concentrated  nitric  acid  are  added. 
This  causes  a  precipitation  of  crystals  of  urea  nitrate. 
Microscopical  examination  reveals  typical  rhomboid  plates 
lying  upon  one  another. 

If  there  is  but  little  fluid,  a  few  drops  of  it  are  placed 
on  a  slide,  a  drop  of  nitric  acid  added,  and  the  slide 
warmed  carefully  over  a  small  flame.  On  cooling,  crystals 
of  urea  nitrate  are  precipitated. 

Pure  urea  may  be  obtained  from  the  urea  nitrate  in 
the  following  manner: 

The  crystalline  precipitate  is  collected  on  a  filter,  dried 
between  two  pieces  of  filter-paper,  and  dissolved  in  a  small 
quantity  of  water.  The  urea  nitrate  is  then  decomposed 
with  barium  carbonate,  the  urea  extracted  from  the  dried 
mass  with  absolute  alcohol,  and  the  liquid  decolorized 
with  animal  charcoal.  When  the  colorless  alcoholic  solu- 
tion, which  has  been  concentrated  by  heating,  cools,  urea 
crystallizes  in  the  form  of  needles.  The  biu ret  reaction  is 
characteristic  of  pure  urea.  A  few  crystals  are  carefully 
heated  in  a  dry  test-tube  until  dissolved.  By  this  pro- 
cess ammonia  and  biuret  (C2H5N302),  which,  when  dis- 
solved in  water,  gives  the  typical  biuret  reaction,  are 
formed.  (For  the  method  of  carrying  out  the  biuret  re- 
action, cf.  p.  149.) 

Uric  Acid:  C5H4N403.— Fifty  to  100  cc  of  urine  are 
rendered  strongly  acid  by  the  addition  of  hydrochloric 
acid.  After  some  hours'  (twelve  to  twenty- four)  stand- 
ing, uric  acid  is  precipitated  in  the  form  of  yellowish- 
brown  crystals.  These  crystals  are  collected  upon  a  small 
filter,  washed  several  times  with  water,  a  portion  placed 
in  a  porcelain  dish,  and  the  murexide  test  carried  out; 
2  to  3  drops  of  concentrated  nitric  a  rid  are  added,  and  the 


132    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

mixture  carefully  heated  over  a  flame  until  the  nitric  acid 
is  driven  off.  The  dry  residue  becomes  violet  upon  the 
addition  of  a  drop  of  sodium  or  potassium  hydrate,  purple- 
red  upon  the  addition  of  ammonia. 

Creatinin:  C4H7N30. — Ten  to  15  cc  of  urine  are  treated 
with  a  few  drops  of  a  dilute  solution  of  sodium  nitro- 
prusside,  and  dilute  sodium  hydrate  is  added  drop  by 
drop.  If  creatinin  is  present,  the  fluid  assumes  a  beauti- 
ful ruby- red  color,  which  remains  but  a  short  time  (in 
dilute  solutions  only  a  few  minutes).  Gradually  the  red 
color  passes  into  a  straw-yellow.  If  this  yellow  fluid  is 
treated  with  glacial  acetic  acid  (5  to  10  drops)  and  heated, 
it  becomes  at  first  green,  then  blue ;  on  longer  standing,  a 
blue  precipitate  is  thrown  down. 

III.  Chemical  and  Physical  Characteristics  of 
the  Urine 

1.  Color. — Normal  urine  may  have  any  shade  of  color 
between  pale  yellow  and  reddish-brown.  Ordinarily,  the 
color  of  the  urine  is  proportional  to  its  concentration. 
Upon  standing  in  the  air,  the  normal  acid  urine  usually 
becomes  darker,  which  is  probably  due  to  the  conversion 
of  the  chromogens  into  pigments,  as  the  result  of  oxida- 
tion. Abnormal  color  of  the  urine  may  be  due  either  to 
pigments  formed  in  the  body,  drugs,  or  articles  of  food. 
Among  the  abnormal  pigments  of  the  urine  are : 

(a)  Blood- Pigment. — The  urine  is  colored  from  pinkish 
red  to  brownish-black. 

(b)  Biliary  Pigments. — The  urine  is  colored  yellowish- 
green  to  dark  brown. 

(c)  Melanin  causes  a  dark  brown  to  black  color.     The 
freshly  passed  urine  contains  only  melanogen,  and  is  not 
deeply  colored.     The  pigment  forms  gradually  upon  stand- 


URINE  133 

ing  in  the  air,  or  upon  the  addition  of  oxidizing  sub- 
stances. 

Drugs  and  articles  of  food  may  cause  the  following 
variations  in  the  color  of  the  urine : 

A  brownish-yellow  to  brownish-black  coloration  follow- 
ing the  internal  or  external  use  of  carbolic  acid,  salicylic 
acid,  preparations  of  cresol,  brenzkatechin,  tar,  folia 
uvse  ursi,  and  similar  preparations,  which  are  excreted  in 
the  urine  in  combination  with  sulphuric  acid. 

Urine  has  a  golden  or  lemon-yellow  color  when  its  reac- 
tion is  acid,  and  a  bright  red  color  when  it  is  alkaline, 
after  the  internal  use  of  rheum,  senna,  cascara  sagrada, 
chrysarobin,  and  similar  preparations  containing  chryso- 
phanic  acid.  Following  the  use  of  santonin,  the  urine  is, 
if  acid,  greenish  or  saffron-yellow  in  color;  if  alkaline, 
red. 

Antipyrin,  sulphonal,  and  trional  cause  a  yellow  to 
blood-red  color. 

Following  the  internal  use  of  methylene  blue,  the  urine 
.  is  colored  blue  or  green. 

2.  Transparency. — The  various  constituents  of  normal 
urine  are  in  solution;  freshly  passed  normal  urine  is,  there- 
fore, perfectly  clear  and  transparent.  However,  a  very 
small  quantity  of  swollen  albuminoid  and  mucoid  sub- 
stances, which  come  from  the  surface  of  the  bladder  and 
the  urethral  mucosa,  are  present,  and  after  standing  a 
short  time,  form  small  clouds,  nubecula,  and  sink  gradu- 
ally to  the  bottom  of  the  vessel. 

If  the  urine  is  cloudy  when  passed,  or  if  it  becomes 
cloudy  soon  after  being  passed,  abnormal  or  pathological 
conditions  may  be  present;  at  any  rate,  the  cause  of  the 
cloudiness  must  be  determined  in  every  individual  case, 
as  it  is  of  the  greatest  importance  for  diagnosis  and  treat- 
ment. 


134    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Cloudy  urine  may  be  due  to  the  following  causes : 

1.  Urinary  salts  suspended  in  the  urine. 

2.  The  presence  of  many  cellular  elements  from  the 
urinary  tract  (blood,  pus-corpuscles,  epithelium). 

8.  Bacteria. 

4.  A  milky  cloudiness  may  be  produced  by  fat  in  emul- 
sion (chyluria  and  lipuria). 

In  order  to  determine  the  cause  of  the  cloudiness  it  is 
well  to  proceed  systematically  as  follows : 

(a)  A  sample  of  the  urine  in  a  test-tube  is  first  care- 
fully warmed  over  a  flame ;  if  the  urine  clears,  the  clouding 
was  due  to  urates.     These  are  often  precipitated  when  the 
urine  has  been  allowed  to  stand,  and  form  the  well-known 
brick-dust  sediment  {sediment um  later itium).      If  this 
uratic  cloudiness  is  accompanied  by  that  due  to  other  sub- 
stances  (usually  cellular  elements),   the  urine  does  not 
become  entirely  clear  when  heated,  but  merely  clears  a 
little,  and  may  even,  if  the  heating  is  continued,  become 
clouded  again  (by  precipitation  of  albumin). 

(b)  If  heating  causes  no  change  in  the  cloudiness, 
10  to  15  drops  of  acetic  acid  are  added;  if  this  renders  the 
urine  entirely  or  partially  clear,  the  cloudiness  was  due 
principally  to  phosphates.     The  urine  often  does  not  be- 
come perfectly  clear,  since  in  such  cases  the  reaction  is, 
as  a  rule,  alkaline,  and  the  urine,  being  somewhat  decom- 
posed, usually  contains,   in  addition,  numerous  bacteria 
or  cellular  elements.     If  acetic  acid  also  exerts  no  influ- 
ence, then: 

(c)  Hydrochloric  acid  is  added.      If  the  cloudiness 
now  disappears,  it  was  due  to  calcium  oxalate. 

(d)  If  the  cloudiness  is  uninfluenced  by  these  three 
procedures,  the  urine  is  first  treated  with  sodium  hydrate 
(10  per  cent.)  and  shaken.     If  in  place  of  the  cloudiness 
a  gelatinous  transparency  appears,  the  cloudiness  was  due 


URINE  135 

to  pus  (Donne's  test  for  pus).  This  test  depends  upon 
the  property  of  pus-corpuscles  to  swell  under  the  influence 
of  alkalies  and  form  a  cohesive  jellylike  mass. 

(e)  If  the  cloudiness  is  due  to  fat,  the  urine  becomes 
entirely  clear  upon  the  addition  of  alcohol  and  ether.  If 
the  cloudiness  resists  all  these  procedures,  it  is,  in  all 
probability,  due  to  bacteria.  In  such  cases  the  urine  is 
evenly  cloudy ;  it  forms  no  noticeable  sediment  upon  stand- 
ing, and  usually  remains  turbid,  even  after  repeated  filtra- 
tion. If  such  urine  is  held  in  a  test-tube  against  the 
light  and  lightly  shaken,  the  cloudiness  is  seen  to  have  a 
fluorescent,  wavy  character. 

3.  Reaction. — The  reaction  of  the  urine  may  be  acid, 
amphoteric,  or  alkaline.  Normal  urine  is  usually  acid  in 
reaction.  Its  acidity  is  not  due  to  the  presence  of  free 
acids,  but  to  salts'  with  acid  reaction,  principally  to  acid 
sodium  phosphate  (mono-sodium  phosphate).  In  addi- 
tion to  the  acid  phosphates,  alkaline  phosphates  are  also 
present  in  the  urine.  Their  quantity  is  usually  small  in 
comparison  with  the  acid  phosphates,  which  explains  the 
acid  reaction. 

If  the  alkaline  phosphates  are  increased,  an  amphoteric 
or  alkaline  reaction  is  produced.  The  reaction  is  called 
amphoteric  when  the  urine  reacts  both  alkaline  and  acid, 
which  is  due  to  the  fact  that  the  phosphates  with  acid 
reaction  and  those  with  alkaline  reaction  are  present  in  the 
urine  in  such  proportions  that  they  exert  an  equal  influ- 
ence upon  its  reaction.  The  reaction  of  the  urine  is  alka- 
line, when  a  large  proportion  of  basic  phosphates  are 
present. 

The  urine  may  be  alkaline,  in  pathological  conditions, 
or  due  to  decomposition  after  standing  in  unclean  recep- 
tacles. Alkaline  or  ammoniaeal  fermentation  of  the  urine 
is  the  conversion  of  urea  into  ammonium  carbonate,  caused 


136    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

by  micro-organisms  (Micrococcus  urece,  Proteus  vulgaris 
ffauseri,  and  others) .  The  urine  becomes  unpleasant  and 
ammoniacal  in  odor  and  cloudy,  due  to  the  precipitation 
of  alkaline  and  earthy  phosphates,  ammonium  magnesium 
phosphate,  ammonium  urate,  and  calcium  carbonate. 

The  reaction  of  the  urine  is  determined  in  the  usual 
manner  with  litmus-paper.  When  the  reaction  is  acid, 
blue  litmus-paper  is  turned  red;  when  alkaline,  red  is 
turned  blue;  and  when  amphoteric,  both  reactions  are 
equally  pronounced.  In  ammoniacal  decomposition  of 
the  urine  the  blue  color  of  the  litmus-paper  disappears 
on  drying  in  the  air.  In  addition,  ammoniacal  urine  is 
characterized  by  the  fact  that,  when  a  glass  rod  wet  with 
hydrochloric  acid  is  held  over  it,  white  clouds  are  formed 
(sal-ammoniac). 

4.  Specific  Grauify.^The  specific  gravity  of  the  urine 
varies  under  physiological  conditions  between  1.005  and 
1.080,  and  depends  principally  upon  the  amount  of  water 
ingested,  and  the  amount  excreted  by  other  organs. 

The  simplest  method  of  determining  the  specific  gravity 
is  by  means  of  two  urinometers,  with  divisions  from  1.000 
to  1.025  and  1.025  to  1.050.  A  rather  wide  glass  cylinder 
is  filled  about  four-fifths  full  of  the  urine  to  be  examined, 
and  the  dried  urinometer  carefully  placed  in  it.1  The 
degree  upon  the  scale  to  which  the  urinometer  sinks  is 
noted.  If  the  urinometer  sinks  too  deeply  (beyond  the 
scale),  or  does  not  sink  to  the  scale,  the  specific  gravity 
must  be  determined  with  the  other  urinometer.  For  exact 
determination  the  temperature  must  be  taken  into  account. 
Urinometers  are  usually  corrected  for  15°  C.  If  the  tem- 
perature of  the  urine  is  higher,  one  degree  (0.001)  must 


1  The  bubbles  of  f oain  which  frequently  form  must  be  removed 
with  filter-paper. 


URINE  137 

be  added  for  each  three  degrees  of  the  thermometer.  If 
the  temperature  is  tinder  15°  C.,  the  same  amount  must 
be  subtracted. 

From  the  specific  gravity  of  the  urine  the  total  solids 
dissolved  in  it  can  be  approximately  calculated.  For  this 
purpose,  according  to  Haeser,  the  last  two  places  of  the 
specific  gravity,  which  is  determined  to  three  decimals, 
are  multiplied  by  0.233.  If,  for  example,  the  specific 
gravity  of  the  urine  is  1.025,  the  percentage  of  solids 
present  is  25X0.233  =  5.825  per  cent. 

5.  The  Freezing- Point  of  the  Urine. — As  early  as  the 
eighteenth  century  (1788)  Blag  den  had  discovered  that  a 
simple  relation  exists  between  the  temperature  at  which 
salt  solutions  freeze  and  the  amount  of  dissolved  matter 
in  the  solutions — namely,  that  the  two  are  proportional. 
This  work  was,  however,  entirely  forgotten.  It  was  only 
after  Raoult  andwm'£  Hoff  had  found  simple  laws  for 
this  fact  that  it  was  turned  to  scientific  use.  These  laws 
are: 

1.  Equimolecular  solutions — i.e. ,  solutions  whose  con- 
centrations are  proportional  to  the  molecular  weights  of 
the  substances  dissolved  have  the  same  freezing-point. 

Example. — The  molecular  weight  of  grape-sugar 
(C6H12O6)  is  180,  that  of  urea  (CON2H4)  60.  A  solution 
which  contains  180  grammes  of  grape-sugar  to  the  litre, 
and  a  solution  which  contains  60  grammes  of  urea  to  the 
litre,  have  the  same  freezing-point.  From  this  it  follows 
that  the  freezing-point  does  not  depend  (as  does  the 
specific  gravity)  upon  the  mass  of  the  substances  dis- 
solved, but  upon  the  number  of  molecules  in  solution,  and 
therefore  can  be  considered  as  a  measure  of  the  molecular 
concentration  of  the  solution. 

2.  The  freezing-point  of  a  solution  is  proportional  to 
its  osmotic  pressure. 


138    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

The  determination  of  the  freezing-point  of  urine  and 
of  blood  was  first  introduced  into  clinical  practice  by  v. 
Koranyij  and  within  a  short  time  has  come  into  compara- 


€— 


E 


FIG.  22 


tively  widespread  use,  especially  in  the  diagnosis  of  func- 
tional kidney  disease. 

For  practical  purposes  the  freezing-point  of  the  urine 


URINE  139 

is  best  determined  by  means  of  Beckmanrfs  apparatus 
(kryoskop) . 1     The  apparatus  is  composed  of  the  following 
parts:  Glass  A  (Fig.  22)  contains  a  thermometer  D,  which 
is  divided  into  0.01  degree,  and  a  stirrer  H  of  bent  plati- 
num wire.     This  glass  is  set  in  another  somewhat  wider 
glass  B,  which  serves  as  an  air-jacket.     The  latter  is  fas- 
tened in  the  cover  of  a  strong  large  jar  C.     In  the  cover 
of  this  large  jar  are  also:  (1)  An  ordinary  thermometer 
for  determining  the  temperature  of  the  freezing-mixture; 
(2)  a  tube  which  contains  a  glass  rod  F.     By  means  of 
the  strong  stirrer  I  the  temperature  of  the  freezing-mixture 
is  kept  even.     The  freezing-point  of  the  urine  is  deter- 
mined in  the  following  manner :  Jar  C  is  filled  about  three- 
quarters  full  of  a  mixture  of  water,  ice,  and  common  salt. 
The  temperature  of  the  freezing-mixture  must  not  be  lower 
than  5°  to  6°  C.     The  urine  to  be  tested  is  placed  in  Glass 
A;  its  quantity  should  be  just  sufficient  to   completely 
cover  the  mercury  reservoir  of  thermometer  D.     In  order 
to  obtain  even  cooling  of  the  urine,  the  freezing-mixture 
and  the  urine  are  constantly  stirred  by  means  of  stirrers 
H  and  I.     The  moment  in  which  the  urine  freezes  is  indi- 
cated by  the  fact  that  the  mercury  column  of  the  ther- 
mometer, which  has  been  up  to  this  time  sinking,  suddenly 
rises  and  remains  at  a  definite  point.     The  rising  of  the 
mercury  column  is  due  to  the  fact  that  before  freezing  the 
fluid  is  always  slightly .  overcooled,  and  the  thermometer 
therefore  falls'  below  the  freezing-point.     As  soon  as  the 
fluid  freezes,  it  is  warmed  to  the  temperature  of  the  freez- 
ing-point, and  therefore  the  mercury  column  rises  to  this 
point.     It  frequently  happens  that,  in  spite  of  marked 

Recently  numerous  simplified  forms  of  apparatus  for  clinical 
use  have  been  suggested.  The  simplifications  have,  however,  un- 
fortunately always  been  made  at  the  expense  of  accuracy. 


140    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

overcooling,  the  fluid  does  not  freeze.  In  such  a  case  a 
small  particle  of  ice  is  introduced  into  the  fluid  through 
tube  G,  by  means  of  rod  F.  The  urine  freezes  at  the 
instant  in  which  the  ice  comes  in  contact  with  it.  Since 
Beclcmann's  thermometer  has  no  constant  zero-point,  the 
latter  must  be  established  by  determining  the  freezing- 
point  of  distilled  water,  and  must  be  frequently  con- 
trolled; this  also  applies  to  those  forms  of  apparatus 
which  have  a  constant  zero-point,  since,  as  experience  has 
shown,  the  zero-point  in  such  forms  of  apparatus  varies 
after  a  time,  and  no  longer  agrees  with  that  marked  upon 
the  scale. 

6.  Quantity. — The  daily  quantity  (twenty-four-hour) 
of  the  urine  is  very  variable,  even  under  physiological 
conditions.  The  ingestion  of  water,  the  excretion  of  water 
by  means  of  the  skin  and  lungs,  the  ingestion  of  diuretic 
substances  '(alcohol,  tea,  coffee),  muscle  activity,  psychic 
and  other  agencies,  influence  greatly  the  excretion  of  urine, 
and  therefore  the  quantity  of  urine  passed  in  twenty- four 
hours.  On  the  average,  the  quantity  passed  by  a  healthy 
man  is  1,500  cc,  by  a  woman  somewhat  less. 

Normally,  the  quantity  of  the  urine  is  in  inverse  pro- 
portion to  its  specific  gravity.  For  estimating  the  daily 
quantity,  the  urine  is  usually  collected  in  a  large  glass 
vessel,  well  shaken,  audits  volume  measured  with  a  gradu- 
ated cylinder.  To  prevent  decomposition  of  the  urine  by 
bacteria,  it  is  well  to  add  to  it  a  piece  of  thymol  the  size 
of  a  bean.  The  weight  of  the  urine  can  be  easily  calcu- 
lated from  its  measured  volume  by  multiplying  the  latter 
by  the  specific  gravity.  For  example,  1,500  cc  of  urine 
of  a  specific  gravity  of  1.025  would  weigh  1,500X1.25  = 
1537.5  grammes. 


URINE  141 

IV.  The  Chemical   Examination  of  the  Pathological 

and  Abnormal  Constituents  of  the  Urine 

1.  Albuminoid  Bodies  in  the  Urine 

Normal  urine  is  free  from  albumin.  The  very  small 
amount  which  can  be  obtained  from  a  large  quantity  of 
urine  does  not  come  into  consideration  in  the  clinical  ex- 
amination, since  it  cannot  be  detected  by  the  usual  tests 
for  albumin.  In  abnormal  and  pathological  conditions 
the  urine  may  contain  the  following  varieties  of  albumin: 

(a)  Serum-albumin. 

(6)  Serum-globulin. 

(<?)  Albumoses  and  peptones. 

(d)  Fibrin. 

(e)  Nucleo-albumin. 

These  albuminoid  substances  are  distinguished  by  the 
following  general  and  physical  characteristics- 

SERUM-ALBUMIN  is  soluble  in  water,  dilute  salt  solu- 
bions,  and  saturated  solutions  of  sodium  chloride  and  mag- 
nesium sulphate.  It  is  precipitated  by  ammonium  sul- 
phate; its  coagulating  temperature  is  72°  to  75°  C  It  is 
precipitated  and  converted  into  acid-albumin  by  concen- 
trated mineral  acids,  but  not  by  dilute;  it  is  converted  by 
alkalies  into  alkali  albumin.  Acid-albumin  is  soluble  in 
acetic  acid. 

SERUM-GLOBULIN. -Insoluble  in  water,  soluble  in  dilute 
lit  solutions,  and  insoluble  in  concentrated  solutions  of 
urn  chloride,  magnesium  sulphate,  and  ammonium 
sulphate.     It  is  coagulated  by  heat  (75°  C. ). 

ALBUMOSES  are  intermediate  products  in  the  hydrolysis 
I  proteids  (the  end-products  are  peptones).     They  cor- 
espondto  propeptone,  and  partially  to  peptone,  as  under- 
stood by  Brmckes.     They  are  not  coagulated  by  heat 


142    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

They  give  a  rose-red  coloration  with  the  biuret  reaction. 
Three  varieties  of  albumoses  are  differentiated  (according 
to  their  solubility). 

(a)  P  rot  albumoses. — Soluble  in  hot  and  cold  water  and 
salt  solutions.     They  are  precipitated  by  saturation  with 
sodium  chloride  and  magnesium  sulphate. 

(b)  Hetero-albumoses. — Insoluble  in  water;  soluble  in 
0.5  to  15  per  cent,  sodium  chloride  solution  at  ordinary 
temperatures.     On  heating  to  65°  C.  they  are  precipitated. 
The  precipitate  is  soluble    in  dilute    alkalies  or   acids. 
Proto-  and  hetero-albumoses  are  frequently  designated  as 
primary  albumoses. 

(c)  Deutero-albumoses  (Secondary  Albumoses) . — Solu- 
ble in  cold  and  hot  water;  are  not  precipitated  by  satura- 
tion with  sodium  chloride  or  magnesium  sulphate,  but  are 
precipitated  by  saturation  with  ammonium  sulphate. 

FIBRIN  is  insoluble  in  water  as  well  as  in  dilute  acids 
and  alkalies.  By  the  latter  it  is  converted. at  ordinary 
temperatures  to  a  jelly,  which  dissolves  after  long  boiling. 

NUCLEO-ALBUMIN  is  insoluble  in  acetic  acid,  and  is 
precipitated  from  solutions  by  it.  Nucleo  albumin  con- 
tains phosphorus. 

2.  Detection  of  Albumin  in  the  Urine 

Two  varieties  of  albuminuria  are  distinguished:  an  ac- 
cidental or  false  albuminuria  (albuminuria  spuria),  and 
a  true  or  renal  albuminuria  (albuminuria  vera).  The 
first  is  due  to  the  admixture  of  fluids  which  contain  albu- 
min, such  as  blood,  pus,  and  chyle,  with  urine,  which, 
when  secreted,  was  free  from  albumin.  Renal  albumi- 
nuria is  caused  by  parenchymatous  changes,  or  disturbances 
of  circulation  in  the  kidney. 

In  both  varieties  of  albuminuria,  the  albumin  in  the 
urine  is  composed  of  a  mixture  of  serum-albumin  and 


URINE  143 

serum-globulin.  Since  these  two  albuminoid  bodies  give 
the  same  reactions,  and  their  division  is  unnecessary  for 
clinical  purposes,  albumin  and  globulin  will  be  estimated 
as  one  in  the  following  tests  for  albumin. 

The  examination  of  the  urine  for  albumin  must  be 
carried  out  with  the  greatest  care,  as  even  the  slightest 
possible  detectable  quantity  of  albumin  has  a  diagnostic 
significance. 

Urine  which  is  to  be  examined  for  albumin  must  be : 

1.  Absolutely  clear. 

2.  Acid  in  reaction. 

3.  Free  from  contamination  with  albuminoid  or  other 
secretions  (menstrual  blood,  sputum)  which  do  not  come 
from  the  urinary  tract 

The  salts,  which  are  suspended  in  the  urine,  bacteria, 
and  cellular  elements  are  removed  by  filtration.  If  the  urine 
is  not  clear  after  filtration,  it  is  treated  with  magnesium 
carbonate,  barium  carbonate,  or  marble-dust,  well  shaken, 
and  again  filtered.  The  substances  producing  the  cloudi- 
ness are  collected  by  the  precipitate,  and  a  clear  filtrate  is 
obtained.  Urine  in  which  the  cloudiness  is  due  to  pre- 
cipitated urates  can  be  most  easily  clarified  by  slight 
heating. 

Of  the  numerous  reactions  recommended  for  the  detec- 
tion of  albumin  in  the  urine,  only  those  will  be  mentioned 
lere  which  have  been  found  in  practice  to  be  reliable  and 
onvenient. 

1.  Heller's  Test  with  Nitric  Acid. — Five  to  ten  cc  of 
trie  acid  are  poured  into  a  test-tube,  or,  what  is  better, 
nto  a  small  conical  glass  (cognac  glass),  which  is  held  at 
n  angle,  and  carefully  covered  with  an  equal  quantity  of 
rine  from  a  pipette.  The  urine  must  be  slowly  and  care- 
ully  floated  upon  the  acid  (it  is  allowed  to  run  from  the 
ipette  along  the  side  of  the  glass)  so  that  the  two  liquids 


144    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

do  not  mix.  If  albumin  is  present,  a  sharply  defined 
annular  cloud  is  formed  on  the  border  between  the  two 
fluids.  When  a  very  small  quantity  of  albumin  is  pres- 
ent, the  ring  is  not  formed  for  two  to  three  minutes. 
The  test  depends  upon  the  ability  of  nitric  acid  to  quickly 
form  acid-albumins,  which  are  with  difficulty  soluble  in 
an  excess  of  acid.  In  this  test  the  following  sources  of 
error  are  to  be  considered. 

(«)  In  very  concentrated  urine  a  distinctly  crystalline 
ring,  composed  of  urea  nitrate,  is  formed  at  the  point  of 
contact  of  the  two  fluids.  With  a  little  care  it  is  very 
easy  to  distinguish  this  distinctly  crystalline  ring  from 
the  opaque,  sharply  defined  albumin-ring.  This  precipita- 
tion of  urea  nitrate  is  prevented  by  diluting  the  urine. 

(b)  In  urine  containing  a  large  amount  of  urates,  an 
annular  cloud  is  also  formed,  which,  however,  is  distin- 
guished from  the  albumin-ring  by  the  fact  that  it  is  situ- 
ated above  the  point  of  contact  (of  the  two  fluids),  and 
disappears  upon  slight  warning. 

(c)  Following  the  internal  use  of  balsamic  prepara- 
tions (copaiba,  balsam  of  Tolu  and  santal  oil),  a  whitish 
ring  is  produced,    due  to  colophonic  acids.     It  is  dis- 
tinguished from  the  albumin-ring  by  the  fact  that  its 
upper  border  is  not  distinct,  and  that  it  is   soluble  in .3 
alcohol. 

(d)  Urine  containing  nucleo-albumin  shows  an  annular 
cloud  with  Heller^s  test.     This  ring,  however,  is  not  sit- 
uated at  the  point  of  contact,  but  approximately  in  the 
middle  of  the  urine  layer;  on  shaking  the  ring  dissolves. 

(e)  Since  nitric  acid  oxidizes  the  urinary  pigments, 
colored  rings    (red,    brown,   blue,   green)   are  formed  in* 
every  urine  during  this  test,  which  can  never  be  confused* 
with  the  albumin-ring,  since  the  cloud  is  absent. 

This  test  gives  a  positive  reaction  with  a  dilution  of 


URINE  145 

1  in  80,000— i.e.,  it  detects  0.0033  per  cent,  albumin. 
With  this  amount  of  albumin  the  ring  is  formed  only 
after  two  minutes.  If  the  above-mentioned  sources  of 
error  are  borne  in  mind  this  test  is  very  accurate  and 
reliable. 

2.  Boiling  with  Sodium  Chloride  and  Acetic  Acid.— 
Five  to  eight  cc  of  urine  are  treated  in  a  test-tube  with  an 
equal  quantity  of  a  saturated  solution  of  sodium  chloride, 
and  after  the  addition  of  3  to  5  drops  of  acetic  acid  boiled. 
If  the  solution  becomes  cloudy,  or  a  precipitate  is  formed, 
albumin  is  present.     In  order  to  detect  very  slight  cloud- 
ing, it  is  well  to  fill  two  test-tubes  with  the  urine  and 
reagent,  and  to  boil  but  one  of  them ;  the  other  is  used  for 
comparison.     If  they  are  both  examined  in  a  good  light 
against  a  dark  background,  the  slightest  clouding  on  boil- 
ing can  be  detected.     When  considerable  albumin  is  pres- 
ent, a  precipitate  is  often  formed  before  heating,  since  the 
albuminoid  bodies  are  converted  by  the  acetic  acid  into 
acid  albumins,  which  are  precipitated  by  the  salt  and  ace- 
tic acid.     Colophonic  acids  are  also  precipitated  by  this 
test,  but  are  dissolved  upon  the  addition  of  alcohol. 

This  test  is  more  delicate  than  Heller's,  and  has  the 
advantage  over  other  boiling  tests  that  the  color  of  the 
urine  remains  unchanged,  and,  therefore,  the  slightest 
cloudiness  can  be  seen,  especially  if  it  is  compared  with 
unboiled  urine  which  has  been  treated  with  the  same 
reagent.  It  is  especially  to  be  recommended  for  the  gen- 
eral practitioner,  since,  in  cases  of  necessity,  it  may  be 
performed  in  a  large  spoon,  without  chemical  apparatus, 
with  ordinary  vinegar  and  common  salt. 

3.  Test  with  Sulpho  salicylic  Acid. — Five  to  ten  cc  of 
filtered  urine  are  treated  with  5  to  10  drops  of  a  20  per 
cent,  solution  of  sulphosalicylic  acid.     In  the  presence  of 
a  small  quantity  of  albumin  the  fluid  becomes  opalescent; 


f 


146    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

in  the  presence  of  a  greater  quantity  a  distinct  cloudiness 
or  a  whitish,  flaky  precipitate  is  produced.  The  urine 
must  always  have  an  acid  reaction ;  alkaline  or  amphoteric 
urine  must  be  slightly  acidified  with  acetic  acid.  Uric 
acid  and  urates  are  not  precipitated  by  this  test.  Albu- 
moses  (with  the  exception  of  deutero-albumoses)  are  pre- 
cipitated, but  they  dissolve  completely  upon  heating, 
while  the  precipitate  of  albumin  remains  unchanged. 
Colophonic  acids  react  with  this  test,  as  with  the  previous 
tests. 

The  test  is  very  delicate;  quantities  of  albumin  from 
0.0015  per  cent,  giving  a  positive  reaction.  To  detect 
slight  ©palescence,  the  urine  is  examined  in  direct  light 
against  a  dark  background,  and  compared  under  the  same 
conditions  with  a  test-tube  containing  an  equal  quantity 
of  clear,  filtered  urine. 

Based  upon  numerous  urine  analyses,  we  can  recom- 
mend this  test  as  both  reliable  and  delicate. 

4.  Test  According  to  Spiegler  and  its  Modification 
According  to  Jolles. 

Spiegler's  reagent  consists  of: 

Hydrarg.  perchlor 8.0 

Acidi  tartarici 4.0 

Glycerini 20.0 

Aquae   dest 200.0 

If  urine  which  has  been  rendered  strongly  acid  with 
acetic  acid  is  floated  upon  this  reagent,  a  whitish  ring  is 
formed  in  the  presence  of  albumin.  This  test  is  very  deli- 
cate, and  detects  even  0.0002  per  cent. ;  it  is,  however, 
unfortunately,  not  always  reliable,  as  the  delicacy  of  the 
reaction  depends  upon  the  quantity  of  chlorides  contained 
in  the  urine.  In  the  presence  of  small  -quantities  of 


URINE  147 

sodium  chloride  the  test  is  not  so  delicate.     To  obviate 
this  the  following  reagent  has  been  suggested  by  Jolles  : 

Hydrarg.  perchlor 10.0 

Acidum  succinicum        .       .       .       .20.0 

Sodium  chlorid 10.0 

Aquae   dest 500.0 

The  reaction  is  carried  out  with  this  reagent  in  the  fol- 
lowing manner:  5  cc  of  urine  are  shaken  with  1  cc  of  30 
per  cent,  acetic  acid  and  4  cc  of  the  reagent.  In  another 
tube  the  same  amount  of  urine  is  shaken  with  1  cc  of  ace- 
tic acid  and  4  cc  of  water.  In  this  second  tube  the  mucin 
will  be  precipitated,  and  by  comparison  albumin  can  be 
detected  in  the  first  tube  if  present. 

The  test  must  be  carried  out  exactly  according  to  the 
instructions;  when  too  little  acetic  acid  is  used  mercury 
combinations  (mercury-phosphorus,  mercury-ammonia) 
may  be  precipitated.  When  urine  contains  iodine,  a 
precipitate  of  iodide  of  mercury  is  formed  with  this  test; 
this  is  soluble  in  alcohol  (in  contradistinction  to  albumin). 

Numerous  tests  have  been  suggested  for  the  detection 
of  albumin  at  the  bedside  which  can  be  carried  out  with 
easily  transportable,  solid  reagents  and  without  boiling. 
These  reagent-tablets  and  reagent-papers  are,  to  be  sure, 
very  convenient,  but  they  are  unreliable,  and  cannot, 
therefore,  be  recommended.  It  is  never  advisable  in  ex- 
amining the  urine  for  albumin  to  limit  one's  self  to  the 
use  of  one  of  the  described  tests,  but  to  carry  out  at  least 
two  of  them.  We  proceed,  as  a  rule,  in  the  following 
manner: 

As  a  preliminary  test  we  use  Heller's  test.  If  this 
yields  a  distinctly  positive  result— i.e.,  if  atypical  al- 
bumin-ring is  formed  at  once— the  urine  contains  a  large 
quantity  of  albumin,  which  is  confirmed  by  .the  boiling 


148    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

test  or  the  sulphosalicylic  acid  test.  If  the  ring  is  formed 
only  after  several  minutes,  merely  a  trace  of  albumin  is 
present,  which  must  be  controlled  by  the  sulphosalicylic 
acid  test,  or  by  boiling  with  sodium  chloride  and  acetic 
acid.  If  with  Hellers  test  no  ring  at  all  is  formed,  the 
urine  may  either  be  entirely  free  from  albumin  or  contain 
only  a  faint  trace.  As  control  tests,  the  most  delicate 
reactions — Jolles*  test  or  the  sulphosalicylic  acid  test — 
must  be  carried  out.  We  recommend  that  the  urine  be 
heated  following  the  sulphosalicylic  acid  test  in  order  that 
there  may  be  no  confusion  with  albumoses. 

3.  Albumoses  and  Peptones 

The  work  of  Stadelmann  and  his  scholars  has  shown 
that  true  peptones,  as  understood  by  Kuelmes^  have  not  as 
yet  been  with  certainty  detected  in  the  urine.  *  All  the 
previous  cases  which  have  been  placed  under  the  head  of 
peptonuria  were,  in  all  probability,  cases  of  the  excretion 
of  albumoses.  These  latter  are  present  in  the  urine  usu- 
ally in  those  diseased  conditions  in  which  there  is  rapid 
destruction  of  normal  or  pathological  tissue ;  in  wide-spread 
exudates  containing  many  cellular  elements,  abscesses, 
and  in  different  febrile  diseases.  When  spermatic  fluid 
is  present  in  the  urine,  traces  of  albumose  are  detected, 
since  this  secretion  contains  albumose.  The  albumoses 
which  appear  in  the  urine  are  composed,  usually,  of  a 
mixture  of  deutero-  and  hetero-albumose.  A  peculiar 
albumose — a  variety  of  hetero-albumose — the  so-called 
Bence-Jones  albumose,  is  present  in  cases  of  multiple 
myeloma.  Very  small  quantities  of  albumose  cannot  be 
detected  with  certainty  in  the  urine;  they  must  be  first 

1  Ito  and  Kotosky  are  said,  recently,  to  have  detected  peptbne- 
Kuehne  in  the  urine.  This  has,  however,  not  yet  been  confirmed  by 
other  authors. 


URINE  149 

isolated  by  precipitation  from  a  large  quantity  of  urine. 
In  urine  rich  in  albumoses  (and  free  from  albumin)  the 
presence  of  the  albumoses  is  shown  by  the  unusual  behavior 
of  the  albumin  tests. 

(a)  With  the  boiling  test,  the  urine  which  has  become 
clear  on  heating,  becomes  cloudy  on  cooling,  and  yields  a 
flaky  precipitate. 

(b)  With  the  sulphosalicylic  acid  test  the  clouding  or 
precipitate  disappears  on  heating  and  returns  on  cooling. 
When  albumin  is  also  present,  the  urine  does  not  become 
entirely  clear  on  heating,   and  must,  therefore,   be  first 
freed  from  albumin  (cf .  below) .     For  the  definite  detection 
of  albumoses  in  the  urine  the  following  method  is  used : 

Ten  cc l  of  urine  are  rendered  acid  with  a  few  drops  of 
hydrochloric  acid,  precipitated  with  phosphotungstic  acid, 
and  centrifugalized;  the  fluid  is  poured  off  from  the  pre- 
cipitate, the  precipitate  well  mixed  with  absolute  alcohol, 
and  again  centrifugalized.  The  washing  with  alcohol  is 
repeated  until  the  alcohol  remains  absolutely  colorless. 
The  precipitate  is  then  dissolved  in  a  small  quantity  of 
sodium  hydrate.  The  now  deep-blue  solution  is  slightly 
heated  until  it  is  again  decolorized.  After  the  solution 
has  cooled,  the  biuret  reaction  is  carried  out — i.e.,  a  very 
dilute  solution  of  copper  sulphate  is  floated  upon  the  alka- 
line liquid.  A  reddish-violet  coloration  is  produced  at 
the  point  of  contact  of  the  two  solutions. 

4.  Method  of  Freeing  the  Urine  from  Albumin 

1.  The  urine  is  treated  with  sodium  acetate  and  with 
enough  ferric  chloride  to  color  it  blood-red.  If  the  urine 
is  markedly  acid,  sodium  hydrate  solution  is  added,  until 
it  becomes  neutral  or  faintly  acid ;  it  is  then  heated  to  the 

1  Urine  containing  albumin  must  first  be  freed  from  the  albumin. 


150    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

boiling-point,  whereupon  the  albumin  is  precipitated  in 
large  flakes. 

2.  The  acid  urine  (neutral  or  alkaline  urine  must  first 
be  slightly  acidified)  is  heated  to  the  boiling-point.  If 
the  albumin  is  not  precipitated  in  large  flakes,  but  the 
solution  merely  becomes  cloudy,  a  few  drops  of  acetic  acid 
are  carefully  added,  and  it  is  heated  a  minute  or  two 
longer.  If  this  does  not  cause  precipitation  of  the  albumin 
in  coarse  flakes,  a  few  cc  of  a  saturated  solution  of  sodium 
chloride  are  added,  and  it  is  again  heated  to  the  boiling- 
point.  The  coagulation  of  the  albumin  depends,  above 
all,  upon  the  quantity  of  acetic  acid  added;  this  must, 
therefore,  be  added  with  great  care.  An  excess  is  as  harm- 
ful as  too  little.  The  filtrate  must  be  clear,  and  must 
show  no  clouding  upon  the  addition  of  sulphosalicylic 
acid  (exception:  urine  containing  albumoses). 

5.  Albumins  which  may  be  Precipitated  by  Cold  Acetic  Acid 

These  albumins  are  mostly  the  nucleo-albumins  and 
the  globulins.  They  are  found  mostly  in  the  physiologi- 
cal, cyclical,  and  orthostatic  albuminurias ;  especially  in 
the  latter.  They  may  be  found  together  with  the  ordinary 
albumin  or  in  a  urine  free  from  albumin.  According  to 
Langstein  they  are  never  or  very  seldom  found  in  the 
urine  of  children  suffering  from  chronic  nephritis,  but,  as 
a  rule  in  orthostatic  albuminuria,  so  that  we  may  use  this 
for  differential  diagnosis.  Not  infrequently  they  occur  in 
the  urine  of  acute  nephritis,  icterus,  and  amyloidosis. 
They  are  tested  for  in  the  following  way : 
To  10  cc  of  the  filtered  urine  are  added  5  to  10  drops 
of  a  80  per  cent,  acetic  acid  solution,  well  shaken  and 
diluted  with  two  or  three  times  its  volume  of  water.  In 
the  presence  of  albumins  which  are  precipitable  by  acetic 
acid,  cloudiness  ensues.  For  the  purpose  of  comparison 


URINE  151 

we  use  another  test-tube  in  which  water  was  added  to  the 
urine  in  like  quantity  and  look  against  a  dark  background. 

6.  Fibrin 

Fibrin  is  either  passed  in  a  coagulated  condition,  or 
separates  out  after  the  urine  has  stood  awhile,  forming  a 
flaky  precipitate.  It  is  insoluble  in  water,  salt  solutions, 
and  in  cold  dilute  acids  and  alkalies.  Fibrin  is  converted 
by  alkalies  into  a  jellylike  mass,  which,  on  continued 
heating,  is  gradually  dissolved;  it  is  also  dissolved  by 
boiling  with  acids. 

For  the  detection  of  fibrin  in  the  urine,  the  suspected 
clot  is  collected  on  a  filter  and  washed  with  a  1  per  cent, 
sodium  chloride  solution  until  the  wash-water  is  no  longer 
alkaline  in  reaction.  The  residue  is  then  digested  and 
dissolved  with  a  warm  5  per  cent,  soda  solution  or  a  0.5 
per  cent,  hydrochloric  acid  solution.  The  solution  must 
give  the  characteristic  albumin  reactions.  The  easiest 
means  of  detecting  fibrin  is  by  its  characteristic  appear- 
ance under  the  microscope. 

THE  CARBOHYDRATES  OF  THE  URINE 

Normal  urine  contains  usually  only  a  very  small  quan- 
tity of  carbohydrates ;  animal  gums  and  a  faint  trace  of 
glucose,  which  cannot  be  detected  by  the  ordinary  reac- 
tions. Under  abnormal  and  pathological  conditions,  in 
addition  to  glucose,  the  following  varieties  of  sugar  may 
be  found :  Lactose,  maltose,  inosite,  and  pentose.  In  the 
clinical  examination  of  the  urine,  glucose  especially  comes 
into  consideration. 

7.  Glucose,  CeHisOe  (Grape-Sugar,  Dextrose) 

The  detection  of  glucose  in  the  urine  depends  upon  the 
following  facts: 


152    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

1.  In  alkaline  solutions  glucose  has  a  tendency  to  ab- 
sorb oxygen  and  therefore  acts  as  a  strong  reducing  agent. 
Metallic  oxides  are  reduced  by  it  to  protoxides  or  to  pure 
metal. 

2.  If  a  solution  of  glucose  is  treated  with  yeast,  alco- 
holic fermentation  takes  place,  by  which  glucose  is  decom- 
posed,   principally   into    alcohol    and   carbon    dioxide, 
C6H1206=2C2H60  +  2C02. 

3.  Glucose  combines  with  phenylhydrazin  in  the  pres- 
ence of  sodium  acetate    forming   a  crystalline  product: 
Phenylglucosazon,  C6H12O6  +  2NH2-NH.  C6H5  =  C6H10O4: 
(N-NHC6H5)2  +  2H20  +  H2. 

4.  Its  watery  solution  rotates  the  plane  of  polarized 
light  to  the  right,  and  in  fact  +52.5°.     Freshly  prepared 
solutions  show  bi-rotation,  which  is  removed  by  heating 
or  long  standing. 

SUGAR  REACTIONS   WHICH  DEPEND    UPON  THE  REDUCING 
PROPERTY  OF  GLUCOSE 

1.  Nylander's  Test. — A  colorless,  alkaline  solution  of 
bismuth  oxide  becomes  black  when  heated  with  glucose, 
since  the  bismuth  oxide  is  converted  into  bismuth  pro- 
toxide or  metallic  bismuth.  Nylander's  reagent  has  the 
following  composition: 

Bismuth  subnitrite 2.0 

Rochelle  salts 4.0 

Sodium  hydrate 10.0 

Aqua  dest 100.0 

Performance  of  the  Test. — Five  to  ten  cc  of  urine  are 
treated  with  15  to  20  drops  (more  does  no  harm)  of  the 
reagent,  and  boiled  two  minutes  (not  less).  In  every 
urine  a  whitish  flaky  precipitate  of  phosphates  is  formed, 


URINE  153 

which,  if  the  urine  contains  no  sugar,  remains  unchanged. 
In  urine  containing  sugar,  first  the  precipitate,  and  then 
the  entire  liquid,  becomes  yellowish-brown  and  finally 
black.  When  small  quantities  of  sugar  are  present  (under 
0. 1  per  cent. )  a  distinct  black  coloration  of  the  precipitate 
cannot  be  seen  during  the  boiling ;  the  coloration  appears 
only  after  the  precipitate  has  settled.  In  such  cases  the 
liquid  is  merely  colored  dark  yellow  or  dark  brown.  The 
solution  must  be  carefully  heated,  in  order  that  it  may 
boil  quietly  without  marked  sputtering.  To  accomplish 
this,  the  test-tube  is  removed  from  the  upper  hot  portion 
of  the  flame  as  soon  as  the  liquid  begins  to  bubble,  and 
held  in  the  cooler  lower  portion  during  the  remainder  of 
the  boiling.  Nylander's  test  is  very  sensitive;  therefore 
when  the  result  is  negative  it  can  be  assumed  with  certainty 
that  the  urine  is  absolutely  free  from  sugar. 

A  positive  result,  however,  does  not  always  mean  the 
presence  of  glucose,  since  other  substances,  which  may  be 
present  in  the  urine,  can  give  the  same  reaction.  The 
most  important  of  these  substances  are  the  following : 

(a)  Albumin. — In  the  presence  of  less  than  0.2  per 
cent,  of  albumin  a  reddish-brown  coloration  is  produced; 
in  the  presence  of  larger  quantities  of  albumin  a  blackish- 
brown  coloration,  which  may  be  confused  with  bismuth, 
reduced  by  sugar.     The  black  coloration  in  urine,  contain- 
ing albumin,  is  due  to*  the  decomposition  of  the  albumin 
and  the  formation  of  bismuth  sulphate.     It  is  well,  there- 
fore, to  free  the  urine  from  albumin,  according  to  one  of 
the  methods    suggested,   before    carrying    out   the  test. 
There  are  also  cases  in  which,  in  spite  of  the  presence  of 
sugar,  reduction  does  not  take  place,  since  the  entire  re- 
agent has  combined  with  albumin.     This,  however,  does 
not  happen  if  sufficient  reagent  is  used. 

(b)  Chrysophanic  acid,  which  is  excreted  in  the  urine 


154    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

following  the  use  of  preparations  of  rheum  and  senna,  also 
reduces  Nylander's  reagent.  Its  presence  is  betrayed  by 
a  reddish  coloration  of  the  urine  upon  the  addition  of  the 
reagent.  This  coloration  resists  the  addition  of  sodium 
hydrate,  but  disappears  absolutely  upon  the  addition  of 
acetic  acid  (in  contradistinction  to  haemoglobin) . 

(c)  Salol,  antipyrin^  menthol,  and  turpentine  oil  are 
excreted  in  the  urine  after  internal  use,  in  combination 
with  glycuronic  acid,  and  faintly  reduce  Nylander's  re- 
agent. 

.  When  the  urine  is  markedly  decomposed,  due  to  fer- 
mentation, and  contains  a  large  quantity  of  ammonium 
carbonate,  the  reaction  may  be  negative  even  in  the  pres- 
ence of  sugar,  since  the  sodium  hydrate  of  the  reagent 
reacts  with  the  ammonium  carbonate,  forming  sodium 
carbonate  and  ammonia;  the  latter  is  driven  off  by  the 
boiling,  so  that  the  strong  alkalinity  necessary  for  the 
reduction  is  no  longer  present. 

2.  Trommer's  Test. — This  depends  upon  the  following 
facts : 

If  copper  sulphate  is  added  to  a  solution  of  sodium 
hydrate,  a  precipitate  of  copper  hydroxide  is  formed, 
which  is  insoluble  in  sodium  hydrate,  2NaOH  +  CuS04  = 
Na2S04  +  Cu(OH2).  This  precipitate  is,  however,  soluble 
in  tartrates,  ammonia,  albumin,  uric  acid,  creatinin,  and 
glucose.  Normal  urine  contains  a  small  quantity  of  some 
of  these  copper  hydroxide  solvents. 

If  an  alkaline  blue  solution  of  copper  hydroxide  is 
heated,  it  remains  unchanged  if  the  solution  contains  no 
reducing  substances. 

If,  however,  such  substances  are  present,  they  form 
copper  protoxide  from  the  copper  hydroxide,  the  liquid 
loses  its  blue  color,  becomes  yellow  or  colorless,  and  a  yel- 
low or  red  precipitate  is  formed.  The  yellow  precipitate 


URINE  155 

consists  of  cuprous  hydroxide,  the  red  of  pure  anhydrous 
cuprous  oxide. 

Procedure. — Five  to  eight  cc  of  urine  are  treated  with 
about  a  quarter  as  much  potassium  or  sodium  hydrate  (10 
per  cent. ) ,  and,  with  vigorous  agitation,  a  10  per  cent, 
solution  of  copper  sulphate  added,  drop  by  drop,  until  a 
small  amount  of  the  copper  hydroxide  which  is  formed, 
remains  undissolved.  The  mixture  is  now  heated,  best  at 
the  surface  of  the  liquid,  to  the  boiling-point.  If  sugar 
is  present,  a  yellow  clouding  is  produced  at  the  portion 
heated,  which,  without  further  heating,  quickly  spreads 
throughout  the  entire  liquid,  and  a  yellow  or  red  finely 
granular  precipitate  is  thrown  down. 

The  reaction  behaves  in  this  manner  only  in  diabetic 
urine. 

When  but  a  small  amount  of  sugar  is  present  (under 
0.5  per  cent.)  the  liquid  turns  yellow,  but  frequently  no 
precipitate  of  cuprous  oxide  is  formed.  On  the  other 
hand,  the  liquid  may  turn  yellow,  and  even  (after  cooling) 
a  late-  precipitation  of  cuprous  oxide  may  be  produced  in 
concentrated  urine,  though  it  is  absolutely  free  from  sugar. 
This  atypical  behavior  of  the  urine  with  Trommels  test 
is  due  to  the  following  facts : 

(a)  The  urine  contains  substances  which  hold  cuprous 
oxide  in  solution  (uric  acid,  creatinin,  ammoniates) . 

(b)  Normal  urine  contains  substances    which  reduce 
cupric  oxide  (uric  acid,  glycuronic  acid,   carbohydrates, 
etc.). 

The  solvent  property  of  normal  urine  for  cuprous  oxide 
is  so  great  that  frequently,  if  normal  urine  is  treated  with 
0.5  per  cent,  glucose  and  Trommels  test  is  carried  out, 
no  precipitation  of  cuprous  oxide  is  produced. 

In  diabetic  urine  the  precipitation  of  cuprous  oxide 
may  occur  in  the  presence  of  less  sugar,  since  the  solvents 


156    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

of  cuprous  oxide  are  relatively  decreased  owing  to  the  poly- 
uria,  which  is,  as  a  rule,  present. 

At  any  rate,  Trommels  test  frequently  leaves  the  ex- 
aminer in  doubt  as  to  the  presence  of  sugar  in  the.  urine, 
and  must,  therefore,  be  considered  as  an  unreliable  and 
inaccurate  test.  It  is  of  value  to  the  practitioner  only  in 
that  in  pronounced  cases  the  quantity  of  sugar  may  be 
roughly  estimated  from  the  volume  of  cuprous  oxide  pre- 
cipitated. For  the  more  exact  qualitative  detection  of 
sugar,  Trammer's  test  should  be  used  only  in  the  follow- 
ing improved  form: 

3.  Fehling's  Test. — Necessary  reagents : 

(Feliling  No.  1.) 

1.  A  7  per  cent,  solution  of  copper  sulphate. 

(Feliling  No.  2.) 

2.  Sodium  hydrate 10.0 

Sodium  potassium  tartrate  (Rochelle  salts)  .  85.0 

Aqua  dest. 100.0 

Procedure. — Ten  drops  of  each  of  these  solutions  are 
placed  in  a  test-tube,  the  mixture  shaken,  diluted  with 
three  times  as  much  water,  and  boiled.  Three  to  five 
drops  of  the  urine  to  be  examined  are  then  added  to  the  hot 
solution,  and  it  is  again  boiled;  if  no  sugar  is  present,  the 
solution  keeps  its  blue  color.  If  considerable  sugar  is 
present,  a  yellow  or  yellowish-red  coloration  of  the  solu- 
tion, and  a  liberal,  finely  granular,  precipitate  of  cuprous 
oxide  are  produced  as  soon  as  the  urine  is  added.  If  less 
sugar  is  present,  the  alteration  of  color  and  the  precipita- 
tion of  cuprous  oxide  do  not  take  place  until  the  solution 
has  been  boiled  again. 

The  solvent  action  of  normal  urine  on  cuprous  oxide 
and  also  its  reducing  power  are  negligible,  owing  to  the 


URINE 


157 


small  amount  of  urine  employed.  By  the  presence  of  the 
sodium  potassium  tartrate  in  the  reagent,  the  precipita- 
tion of  cupric  hydroxide  which,  on  boiling,  forms  black 
oxide  of  copper  and  often  obscures  the  reaction  in  Trom- 
mels test,  is  entirely  prevented,  since  the  Rochelle  salts 
hold  the  cupric  hydroxide  in  solution. 

4.  Fermentation    Test.— A.   piece  of    fresh  compressed 
yeast,   the  size  of  a  pea,  which  must  be  absolutely  free 


FIG.  23 

from  sugar,  is  rubbed  in  a  test-tube  with  a  small  quantity 

of  urine  (1  to  2  cc),  and  20  to  25  cc  of  urine  are  added  to 

is  paste.     If  the  urine  is  alkaline  in  reaction,  it  is  acidi- 

fcd  with  tartaric  acid.     Einhorn^s  saccharometer   (Fig 

is  filled  with  this  urine  in  such  a  manner  that  tube  a 


158    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

is  completely  filled,  to  the  entire  exclusion  of  air,  and 
bulb  c  half -filled,  and  is  set  aside  in  a  moderately  warm 
place  (25  to  30°  C.).  The  horizontal  portion  of  the 
apparatus  Z>  may  be  closed  with  a  few  drops  of  mercury, 
but  this  is  not  necessary  for  the  qualitative  test.  In  the 
presence  of  sugar,  even  after  a  few  hours,  carbon  dioxide 
collects  at  the  top  of  tube  a,  since  the  sugar  is  decom- 
posed by  fermentation  into  alcohol  and  carbon  dioxide. 
If  sugar  is  absent,  no  gas  is  formed.  The  test  should  not 
be  considered  as  completed  before  the  end  of  twenty- four 
hours.  As  commercial  yeast  is  not  always  free  from  sugar 
and  may,  therefore,  be  fermentable,  two  controls  should 
be  made  at  the  same  time :  one  saccharometer  should  be 
filled,  in  the  manner  described,  with  water  acidified  with 
tartaric  acid  and  yeast;  the  other  with  an  acidified  glucose 
solution  (0.5  per  cent.)  and  yeast.  The  fitness  of  the 
yeast  for  use  is  established,  if  no  gas  forms  in  the  first 
control  saccharometer,  while  a  large  amount  of  carbon 
dioxide  forms  in  the  second.  If  proof  is  desired  that  the 
gas  produced  is  really  carbon  dioxide,  a  small  amount  of 
sodium  hydrate  solution  may  be  introduced  into  tube  a  by 
means  of  a  curved  pipette.  If  the  gas  bubble  disappears, 
it  was  composed  of  carbon  dioxide.  The  fermentation 
test  is  the  only  absolutely  sure  and  exact  method  for  the 
detection  of  sugar  in  the  urine,  since  no  other  normal  or 
pathological  constituent  of  the  urine  gives  a  similar  reac- 
tion. It  is  at  the  same  time  sufficiently  delicate.  The 
presence  of  sugar,  from  0.05  per  cent,  on,  is  distinctly 
detected  by  means  of  this  test. 

5.  The  Phenyl-Hydrazin  Test  (According  to  Koivarsky). — 
Five  drops  of  pure  phenyl-hydrazin  (phenyl-liydrazinum 
purum)  are  treated  in  an  ordinary  test-tube  with  10  drops 
of  acetic  acid  and  the  mixture  lightly  shaken.  About  15 
drops  of  a  saturated  solution  of  sodium  chloride  are  then 


URINE  159 

added,  whereupon  the  mixture  congeals  to  a  paste.  About 
10  cc  of  urine  are  added  to  this  paste,  and  heated  carefully 
over  a  flame.  The  solution  is  boiled  at  least  two  minutes. 
On  slowly  cooling,  a  yellow  precipitate,  consisting  of  typical 
crystals  of  phenyl-glucosazone,  is  thrown  down.  The 
rapidity  of  the  precipitation  depends  upon  the  amount  of 
sugar  in  the  urine.  In  the  presence  of  more  than  0.2  per 
cent,  of  sugar  the  precipitation  is  formed  in  a  few  min- 
utes; in  the  presence  of  less,  often  not  for  five  minutes  to 
half  an  hour.  This  test  is  very  delicate  and  detects  sugar 
from  0.08  per  cent,  onward. 

Opinions  are  as  yet  divided  as  to  the  practical  value 
of  the  phenyl-hydrazin  test;  on  the  one  hand,  it  is  claimed 
that  normal  urine  contains  substances  which  combine  with 
phenyl-hydrazin  to  form  osazone  and  produce  similar 
crystals;  on  the  other  hand,  it  is  considered  as  reliable, 
and  as  furnishing  positive  evidence  in  doubtful  cases. 

The  fact  is  that  normal  urine  frequently  contains  sub- 
stances (combinations  with  glycuronic  acid)  which  pro- 
duce similar  crystals  with  this  test.  These  substances  are, 
however,  present  in  very  small  quantity,  and  with  a  cer- 
tain amount  of  practice  are  easy  to  distinguish  from  the 
typical  glucosazone  crystals,  since  they  are  plumper  and 
thicker  than  the  true  crystals,  and  not  so  typically  arranged. 
In  addition  to  the  glycuronic  acid  compounds  the  pentoses 
also  form  osazone.  As  yet,  however,  but  a  few  cases  of 
true  pentosuria  have  been  reported  in  the  entire  literature, 
BO  that  these  carbohydrates  are  of  little  importance  in  con- 
sidering the  phenyl-hydrazin  test.  Based  upon  our  own 
experience,  which  covers  many  thousands  of  urine  analy- 
ses (in  which  the  result  of  the  phenyl-hydrazin  test  has 
always  been  controlled  by  the  fermentation  test),  we  con- 
'sider  this  test  very  delicate  and  reliable,  and  can  recom- 
mend it  especially  in  doubtful  cases. 


160    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Concerning  the  detection  of  sugar  by  polarization,  cf  . 
the  Quantitative  Examination  of  the  Urine. 

8.  Lactose,  Ci2H22Oii  (Milk-Sugar) 

Lactose  is  present  in  small  amount  (maximum  1  per 
cent.  )  in  the  urine  of  women  when  there  is  stagnation  of 
milk.  Like  glucose,  it  possesses  the  property  of  reducing 
metallic  oxides  in  alkaline  solution,  and  of  rotating  the 
plane  of  polarized  light  to  the  right.  It  is  not,  however, 
subject  to  alcoholic  fermentation  by  yeast.  On  boiling 
with  dilute  acids  it  is  converted  into  glucose  and  ferment- 
able galactose.  The  latter  also  forms  a  crystalline  combi- 
nation with  phenyl-hydrazin  (galactosazone).  According 
to  Riibner,  lactose  is  detected  in  the  urine  by  the  follow- 
ing test  : 

Ten  cc  of  urine  are  boiled  three  to  four  minutes  with 
a  large  quantity  of  lead  acetate  ;  in  the  presence  of  lactose 
the  solution  becomes  yellow  or  brown  ;  ammonia  is  added 
to  the  hot  solution  as  long  as  the  precipitate  is  dissolved. 
The  solution  first  assumes,  an  intense  brick-  red  color,  then 
throws  down  a  beautiful  cherry-red  to  copper-  colored  pre- 
cipitate, and  becomes  colorless.  The  reaction  is  not  deli- 
cate, and  only  detects  positively  0.3  to  0.5  per  cent,  or 
more  of  lactose.  If  it  is  of  especial  importance  to  detect 
lactose,  it  must  be  isolated  from  a  large  quantity  of  urine 
and  tested  in  its  pure  state. 


9.  Levulose,  CeH^Oe  (Fruit-Sugar) 

Levulose  rarely  appears  in  the  urine,  but  when  present 
is  usually  accompanied  by  glucose.  It  differs  from  glucose 
in  that  it  rotates  the  plane  of  polarized  light  to  the  left. 
Its  behavior  toward  the  metallic  oxides,  yeast,  and  phenyl- 
hydrazin  is  identical  with  that  of  glucose.  For  the  detec- 
tion of  levulose  in  the  urine  the  sugar  present  must  be 


,     URINE  161 

estimated  by  at  least  two  methods — by  polarization  and 
fermentation,  or  by  polarization  and  titration  according 
to  Fehling.  Levulose  is  detected  if  the  urine  rotates  the 
plane  of  polarized  light  less  to  the  right  than  corresponds 
to  the  amount  of  sugar  present,  estimated  by  another  ex- 
act quantitative  method.  It  must,  however,  also  be  deter- 
mined that  there  are  no  other  laevorotary  substances 
(albumin,  /?-oxy butyric  acid,  etc.)  present  in  the  urine. 

Seliwanoff  has  suggested  the  following  color  reaction 
for  the  detection  of  levulose:  A  solution  of  levulose  is 
heated  with  resorcin  and  hydrochloric  acid,  a  precipitate 
is  formed  which  dissolves  in  alcohol  with  the  production 
of  a  red  color.  This  reaction  is  not  very  accurate.1  For 
positive  determination  levulose  must  (like  lactose)  be  iso- 
lated and  tested  in  its  pure  state. 

10.  Pentose,  C5HioO5  (Pentaglucose) 

Pentoses  differ  from  other  varieties  of  sugar  in  that 
they  are  unfermentable.  They  reduce  Fehling1  s  solution 
only  after  long  heating,  and  only  very  faintly  reduce 
Nylander^s  reagent.  Pentoses  are  best  detected  in  the 
urine  by  means  of  the  orcin  test,  which  is  carried  out  in 
the  following  manner : 

Five  to  eight  cc  of  fuming  hydrochloric  acid  are  slightly 
supersaturated,  under  heating,  with  orcin  (a  knife-point 
of  orcin  is  sufficient) .  One  to  two  cc  of  urine  are  added 
to  the  hot  solution,  and  it  is  again  heated  to  the  boiling- 
point.  If  pentoses  are  present  the  solution  turns  green. 
The  pigment  is  extracted  with  a  small  amount  of  amyl 
alcohol  and  examined  spectroscopically.  An  absorption 
band  is  seen  in  red  between  C  and  D.  The  orcin  test  de- 
pends upon  the  formation  of  furfurol,  which  when  boiled 
with  orcin  and  hydrochloric  acid  produces  a  green  pigment. 

1  According  to  Loew  pyrogallol  gives  a  similar  reaction. 


162     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

11.  Glycuronic  Acid  (CHO  [CHOH]4  COOH) 

According  to  its  chemical  composition  glycuronic  acid 
is  closely  related  to  the  carbohydrates.  It  is  considered 
as  the  first  product  of  the  oxidation  of  glucose.  Free 
glycuronic  acid  does  not  appear  in  the  urine;  it  is  ex- 
creted usually  as  a  double  compound  with  phenyl,  skatol, 
indoxyl,  thymol,  etc.,  both  in  normal  and  pathological 
urine.  It  comes  into  consideration  in  the  clinical  exami- 
nation of  urine  only  in  that  it  gives  certain  reactions  which 
resemble  those  of  glucose,  and  may  often  be  confused  with 
it.  Free  glycuronic  acid  rotates  the  plane  of  polarized 
light  to  the  right,  combined  glycuronic  acid  to  the  left. 
The  slight  laevorotary  action  of  normal  urine  is  in  all  prob- 
ability due  to  the  presence  of  glycuronic  acid.  This  may 
be  established  by  the  fact  that  after  boiling  with  dilute 
acids  (sulphuric  acid)  the  urine  shows  a  dextrorotary 
action,  since  glycuronic  acid  is  freed  by  boiling  with  acids. 
It  slightly  reduces  Feliling's  and  Nylander's  reagents. 
Glycuronic  acid  compounds  are  precipitated  by  lead  acetate 
(in  contradistinction  to  glucose).  The  reducing  action 
and  the  laBvorotary  action  are  not,  however,  positive  proof 
of  the  presence  of  combined  glycuronic  acid.  For  the 
positive  detection  of  combined  glycuronic  acid  the  follow- 
ing procedure  must,  according  to  ScdJcowsJci  and  Paul 
Mayer,  be  carried  out.  The  phloroglucin  test  is  first 
carried  out. 

Five  to  six  cc  of  fuming  hydrochloric  acid  are  satura- 
ted with  phloroglucin  while  hot,  so  that  on  cooling  a  small 
excess  of  the  latter  remains  undissolved,  and  this  solution 
is  divided  into  two  portions,  to  one  of  which,  after  cooling, 
J  cc  of  the  urine  to  be  examined  is  added,  and  to  the  other 
the  same  amount  of  normal  urine  of  nearly  equal  concen- 
tration. If  both  tubes  are  plunged  into  a  beaker  of  boil- 


URINE  163 

ing  water,  the  urine  containing  glycuronic  acid  assumes 
an  intense  red  color,  which  gradually  spreads  from  above 
downward.  The  normal  urine  does  not  change  its  color, 
or  changes  it  very  slightly.  As  pentoses  also  give  this 
reaction,  it  must  be  determined  that  an  untreated  sample 
of  the  urine  gives  a  negative  result  with  the  orcin  test. 
This  latter  reaction  must,  however,  be  distinctly  given, 
after  boiling  with  acids,  by  urine  containing  combined 
glycuronic  acid. 

Fifty  cc  of  urine  are  treated  with  sufficient  concentrated 
sulphuric  acid  to  make  the  solution  correspond  to  a  1  per 
cent,  solution  of  sulphuric  acid,  and  heated  in  a  porcelain 
dish  over  a  free  flame.  The  exact  length  of  heating  can- 
not be  given.  It  is  usually  sufficient  to  keep  the  urine 
boiling  for  one  to  three  minutes.  The  orcin  test  is  then 
carried  out  directly  with  the  solution  without  filtering  it. 
If  the  reaction  does  not  take  place  at  once,  the  solution 
must  be  again  boiled  for  one  to  two  minutes. 

12.  Acetone,  Diacetic  Acid,  /?-Oxybutyric  Acid 

ACETONE,  CH3.CO.CH3  . 

Normal  urine  contains  but  a  very  faint  trace  of  ace- 
tone, too  slight  to  be  detected  by  the  ordinary  reactions 
(at  the  most  0.01  gramme  in  the  daily  output).  Under 
abnormal  and  pathological  conditions  (diabetes,  fever, 
exclusive  meat  diet,  starvation,  disturbances  of  digestion) 
the  amount  of  acetone  in  the  urine  may  reach  0.5  or  even 
1.0  gramme  in  twenty-four  hours. 

Detection. — Legal* s  Test. — Eight  to  ten  cc  of  urine  are 
treated  with  8  to  5  drops  of  a  freshly  prepared  saturated 
solution  of  sodium  nitroprusside,  and  rendered  alkaline 
with  a  few  drops  of  sodium  hydrate.  On  the  addition  of 
the  sodium  hydrate,  a  ruby- red  coloration  appears  in  almost 


164    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

every  urine,  and  is  due  to  a  normal  constituent  of  the 
urine,  creatinin.  If  the  red  solution  is  supersaturated 
with  concentrated  acetic  acid,  the  red  color  becomes  more 
intense  in  the  presence  of  acetone,  and  passes  into  crim- 
son, while  if  acetone  is  not  present  the  red  color  entirely 
disappears.  The  reaction  is  not  very  delicate :  according 
to  von  Jakscli,  only  quantities  over  0.8  milligramme  are 
detected.  To  detect  smaller  quantities  the  acidified  urine 
(100  cm3)  must  be  distilled  and  the  first  portion  of  the 
distillate  must  be  examined  with  the  more  delicate. 

Lichen's  lodoform  Test. — Five  to  ten  cc  of  the  distillate 
are  treated  with  LugoVs  iodine  potassium  iodide  solution, 
and  sodium  hydrate.  In  the  presence  of  acetone,  iodoform 
is  produced  which  may  be  easily  recognized  by  its  odor 
and  its  crystalline  forms  (hexagonal  and  stellate  plates). 
This  reaction  is  much  more  delicate  than  LegaVs;  it  has, 
however,  the  disadvantage  that  alcohol  and  aldehyde  also 
give  this  reaction,  and  alcohol  (due  to  fermentation  of 
the  sugar)  may  be  present  in  precisely  those  cases  in  which 
the  detection  of  acetone  is  of  the  greatest  importance — 
namely,  in  diabetic  urine. 

DIACETIC  ACID,  CH3COCH2COOH 

Diacetic  acid  is  almost  never  present  in  the  urine,  ex- 
cept under  pathological  conditions.  It  is  very  frequently 
accompanied  by  acetone  and  /8-oxybutyric  acid.  It  is 
formed  from  /?-oxy butyric  acid,  and  decomposes  easily 
into  acetone  and  carbon  dioxide.  The  urine  must,  there- 
fore, be  examined  in  as  fresh  a  condition  as  possible. 

Detection. — Gerhard? s  Test. — Five  to  ten  cc  of  urine 
are  treated  with  a  solution  of  ferric  chloride  as  long  as  a 
sediment  is  formed.  In  the  presence  of  diacetic  acid  a 
Bordeaux-red  coloration  is  produced.  If  the  red  color  of 
the  solution  is  not  distinct,  it  is  well  to  remove  the  pre- 


URINE  165 

cipitate  of  ferric  phosphate  by  filtration.  The  urine  gives 
a  very  similar  reaction  following  the  internal  use  of  sali- 
cylic acid,  antipyrin,  thallin,  phenacetin,  and  certain 
other  drugs.  A  positive  result  of  the  reaction  must,  there- 
fore, be  confirmed  by  a  control  test.  Five  to  ten  cc  of  the 
urine  are  boiled  three  to  five  minutes,  and,  after  cooling, 
are  treated  with  ferric  chloride  in  the  above-described 
manner.  If  the  positive  result  was  due  to  diacetic  acid 
the  red  color  will  not  appear,  since  diacetic  acid  is  decom- 
posed by  boiling;  if  it  was  due  to  drugs,  the  red  color, 
upon  the  addition  of  ferric  chloride,  will  appear  after  boil- 
ing as  well  as  before. 

£-OXYBUTYEIC  ACID,  CH3CH  (OH)  CH2COOH 

This  acid  appears  in  the  urine  in  severe  cases  of  dia- 
betes and  is  always  accompanied  by  acetone  and  diacetic 
acid,  which  are  considered  as  products  of  its  decomposi- 
tion. It  rotates  the  plane  of  polarized  light  to  the  left, 
and  can,  therefore,  influence  the  quantitative  estimation 
of  glucose  by  polarization,  or  even  render  it  impossible. 

According  to  Kuelz,  /?-oxybutyric  acid  is  detected  in 
the  urine  in  the  following  manner : 

The  urine  is  fermented  with  yeast,  and  most  of  the 
laevorotary  substances — with  the  exception  of  /?-oxybutyric 
acid — are  precipitated  with  lead  acetate  and  ammonia. 
The  filtrate  is  examined  with  the  polarimeter.  Rotation 
to  the  left  suggests  the  presence  of  the  acid. 

13.  The  Pigments  and  Chromogens  of  the  Urine 

A.  INDICAN   (INDOXYLSULPHURIC  ACID), 
C8H6NOSO2OH 

Normal  human  urine  contains  but  a  small  quantity  of 
indican — on  the  average,  0.06  gramme  in  twenty- four 
hours.  Under  pathological  conditions,  ten  to  fifteen  times 


166    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

this  quantity  may  be  present.  Indol,  from  which  indican 
is  derived,  is  formed  in  the  intestines  as  a  product  of  the 
decomposition  of  albumin.  After  its  absorption  indol  is 
oxidized  to  indoxyl,  and  then  combines  with  the  sulphuric 
acid  of  the  urine,  and  is  thus  excreted  as  indoxylsulphuric 
acid.  Indican  in  the  urine  may  .be  decomposed  by  min- 
eral acids  and  the  indoxyl  converted  by  oxidation  into 
indigo.  Under  pathological  conditions  there  may  be  a 
spontaneous  excretion  of  indigo  in  the  urine,  in  which 
case  it  may  be  either  in  solution  or  in  the  sediment. 

Detection. — One-third  of  a  test-tube  of  urine  is  treated 
with  an  equal  quantity  of  concentrated  hydrochloric  acid, 
15  drops  of  chloroform,  and  2  to  8  drops  of  a  2  per  cent, 
potassium  permanganate  solution.  The  test-tube  is  corked 
and  repeatedly  inverted,  whereupon  the  indigo-blue  which 
is  formed  is  extracted  by  the  chloroform,  coloring  the 
latter  distinctly  blue.  It  must  not  be  vigorously  shaken, 
since  chloroform  forms  an  emulsion  with  the  urine  which 
it  is  very  hard  to  decompose. 

The  reaction  depends  upon  the  decomposition  of  the 
indican  by  hydrochloric  acid,  and  the  oxidation  of  the 
freed  indoxyl,  by  potassium  permanganate  to  indigo-blue. 
The  potassium  permanganate  must  be  added  very  care- 
fully; at  first,  not  more  than  2  to  3  drops  are  added,  since 
by  too  strong  oxidation  the  indigo-blue  can  be  at  once 
further  oxidized  to  yellow  isatin,  and  thus  be  entirely 
overlooked.  If  the  permanganate  solution  is  further 
added  a  drop  at  a  time,  it  will  be  noticed  that  in  urine 
containing  but  little  indican  the  blue  coloration  of  the 
chloroform  disappears  after  but  a  few  drops,  while  iix 
urine  rich  in  indican  the  blue  coloration  becomes  more 
intense  as  the  solution  is  added,  and  a  comparatively 
large  amount  of  the  solution  must  be  added  before  the 
indigo-blue  is  entirely  converted  into  isatin,  This  pro- 


URINE  167 

cedure  may  be  used  as  a  method  for  the  quantitative 
estimation  of  indican. 

Not  infrequently  in  the  indican  test  a  rose-red  coloration 
of  the  chloroform  is  produced,  instead  of  a  blue  coloration. 
This  is  the  case  following  the  internal  use  of  preparations 
of  iodine.  The  iodine  is  freed  from  its  combinations  by 
the  hydrochloric  acid  and  the  oxidizing  material,  and 
causes  the  red  coloration  of  the  chloroform.  To  offset  this 
a  crystal  of  sodium  thiosulphate  is  added,  and  the  solu- 
tion shaken.  The  iodine  then  forms  colorless  iodides, 
and  the  chloroform  is  decolorized.  In  the  presence  of  both 
iodine  and  indican  a  violet  coloration  of  the  chloroform  is 
produced,  which,  on  treating  with  sodium  thiosulphate, 
becomes  blue. 

In  the  presence  of  chrysophanic  acid  a  greenish-yellow 
coloration  of  the  chloroform  is  produced  with  the  indican 
test. 

A  yellowish  coloration  is  produced  following  the 
internal  use  of  bromine  preparations. 

B.  UROBILIN  AND  UROBILINOGEN 

Normal  urine  contains  a  very  small  quantity  of  urobilin 
in  the  form  of  a  chromogen — urobilinogen — which  by  the 
action  of  light,  and  by  the  presence  of  acids,  is  very  easily 
converted  into  the  pigment.  In  its  pure  state  urobilin 
is  an  amorphous,  reddish-brown  substance,  soluble  with 
difficulty  in  water.  It  is  readily  soluble  in  alcohol,  chloro- 
form, and  alkalies.  It  forms  insoluble  salts  with  the 
alkaline  earths  and  the  heavy  metals. 

Detection  by  the  Schlesinger  Method. — Ten  to  fifteen  cc  of 
urine  are  treated  with  an  equal  quantity  of  10  per  cent, 
alcohol  solution  of  zinc  acetate,  and  filtered.  The  filtrate 
when  held  against  a  dark  background  shows  a  distinct 
greenish  fluorescence.  This  test  is  very  delicate, 


168    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

The  test  for  Urobilinogen  by  Elirlicli's  aldehyd  reaction. 

A  few  drops  of  a  2  per  cent,  solution  of  dimethyl- 
paramido-benz-aldehyd  in  20  per  cent.  HC1  are  added  to 
a  few  cc  of  urine  and,  should  the  reaction  not  appear  soon, 
a  little  HC1  concentrated  is  added.  Most  urines  are  col- 
ored slightly  red  by  this.  In  the  presence  of  an  increased 
quantity  of  urobilinogen  the  red  color  becomes  quite  in- 
tense and  is  clearly  demonstrable  even  after  several  dilu- 
tions; spectroscopic  analysis  shows  a  broad  absorption 
band  between  Frauenhofer'' s  lines  D  and  E.  Urobilin  does 
not  give  this  reaction. 

C.  BILIARY  PIGMENTS 

Normal  urine  contains  no  biliary  pigments.  Under 
pathological  conditions  these  gain  entrance  to  the  circula- 
tion, and  thence  to  the  urine.  Bilirubin  is  the  only  one 
of  the  biliary  pigments  which  has  with  certainty  been  de- 
tected in  fresh  icteric  urine.  The  others — biliverdin, 
biliprasin,  bilifuscin — are  formed  only  after  the  urine  has 
stood  a  long  time,  and  must  therefore  be  considered  as 
derivatives  of  bilirubin. 

Icteric  urine  is  saffron-yellow  to  reddish  or  dark-brown 
in  color.  On  shaking,  a  characteristic  yellow  foam  is  pro- 
duced. 

Detection. —  (a)  Gmelin's  Test. — Five  to  six  cc  of  ordi- 
nary concentrated  nitric  acid  are  treated  with  a  few  drops 
of  yellow,  fuming  nitric  acid,  and  carefully  covered  with 
an  equal  quantity  of  the  urine  to  be  examined.  In  the 
presence  of  biliary  pigments  an  emerald-green  ring  is 
formed  at  the  point  of  contact  of  the  two  liquids,  beneath 
which  a  blue,  violet,  or  yellow  ring  is  gradually  formed. 
The  test  should  be  considered  positive  only  when  the  green 
ring  is  very  pronounced,  since  the  blue  and  violet  rings 
may  be  caused  by  the  oxidation  of  other  substances 


URINE  169 

which  appear  in  the  urine  (indican,  indigo  red).  This 
test  is  not  delicate;  it  detects  the  presence  of  only  5  per 
cent,  or  more  of  bile. 

(b)  Modification  According    to    Rosenbach. — A  large 
quantity  of  urine  is  filtered  through  a  single  filter-paper, 
and  the  inner  side  of  the  paper  is  touched  with  a  drop  of 
nitric  acid  which  contains  nitrous  acid  (prepare  as  in  test 
a) .     The  colors  are  then  produced  as  in  test  a. 

This  test  is  somewhat  more  delicate  than  Gmelin'stest, 
and  is  more  distinct  in  the  presence  of  small  amounts  of 
bile-pigment.  It  must  be  remembered,  however,  in  this 
test  that  a  green  ring  may  be  produced  following  the  use 
of  antipyrin.  Not  rarely  a  blue  color  is  produced,  follow- 
ing the  ingestionof  preparations  of  iodine,  since  the  iodine 
is  freed  by  the  nitric  and  nitrous  acids,  and,  combining 
with  the  starch  present  in  the  filter-paper,  causes  a  more 
or  less  intense  blue  coloration.  This  blue  color  may  abso- 
lutely obscure  the  green  color  of  the  bile-pigment. 

(c)  Test  with  Tincture  of  Iodine  According  to  Rosin. 
—The  urine  (10  to  15  cc)  is  covered  in  a  test-tube  with  a 

dilution  of  1:10  of  the  official  tincture  of  iodine.  A  green 
ring,  which  lasts  for  hours,  is  formed  at  the  point  of  con- 
tact of  the  solutions.  The  test  is  more  delicate  than 
Gmeliri's. 

(d)  Huppertfs    Test. — One  hundred  cc  of  urine  are 
rendered  distinctly  alkaline  with  sodium  carbonate,  and 
the  biliary   pigments  are  precipitated  with  an  excess  of 
barium   chloride   or   barium   hydrate.     The   yellow  pre- 
cipitate is  collected  on  a  filter,  and  boiled  with  alcohol, 
to  which  a  few  drops  of  diluted  sulphuric  acid  have  been 
added.     The  precipitate  is  decolorized  by  this  procedure, 
while  the  solution  assumes  a  beautiful  green  color.     After 
this   alcoholic   solution  has  been  diluted  with  an  equal 
quantity  of  water,  the  pigment  can  be  extracted  with  a 


170    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

few  cc  of  chloroform.  The  chloroform  becomes  deep 
green.  This  is  the  surest  and  most  delicate  method  for 
detecting  biliary  pigments  in  the  urine. 

D.  BLOOD  PIGMENT,  HEMOGLOBIN 

The  following   varieties    of    hemoglobin  are  distin- 
guished : 

1.  Oxyhsemoglobin. 

2.  Methsemoglobin,  contains  the  same  amount  of  oxy- 
gen as  oxyhsemoglobin,  but  in  more  stable  combination. 


Oxy  haemoglobin. 

Methaemoglobin. 

Reduced  Haemoglobin. 

Soluble    in    water, 

Solutions   brown  in 

In  moderately  dilute 

coloring  it  bright- 

color  ;  crystallizes 

solutions,    green- 

red; insoluble  in 

in    the     form    of 

ish   to   brownish- 

alcohol  ;  crystalli- 

brown  needles  and 

red  ;     is     formed 

zable  ;  easily  de- 

plates;   is   easily 

from     oxyhaemo- 

composed  in   wa- 

formed from  oxy- 

globin  and  methae- 

tery  solu  ti  ons. 

haemoglobin      by 

moglobin   by   the 

On  heating  these 

the  action  of  acids 

action  of  reducing 

solutions,  abrown- 

and    acid     salts  ; 

substances  ;      for 

ish  precipitate  is 

hence  its  presence 

example,    by   the 

formed  even  at  70° 

in  the   urine.     In 

addition  of  a  few 

C.  ,  which  is  com- 

acid or  neutral  so- 

drops of  ammoni- 

posed of  albumin 

lutions,  it  shows, 

um  hydrosulphide 

andhsematin.  Di- 

in addition  to  the 

or  stannous  chlo- 

lute solutions  are 

absorption   bands 

ride  in  ammonia- 

yellowish-red     in 

of     oxyhaemoglo- 

cal     solution. 

color,    and    show 

bin,    two    bands, 

Shows    only    one 

spectroscopically 

the  first  of  which 

broad    absorption 

(0.01    per   cent.) 

is     more      pro- 

band. On  shaking 

characteristic  ab- 

nounced than  the 

with  air,  it  is  con- 

sorption '   bands. 

others. 

verted  into    oxy- 

It  is  also  decom- 

haemoglobin; with 

posed  into  haema- 

acetic  acid  and  a 

tin    and    albumin 

trace    of   sodium 

by  the   action  of 

chloride,  it  forms 

acids  and  alkalies. 

haemin   (haematin 

chloride,       dark- 

brown     rhomboid 

plates). 

URINE  171 

8.  Reduced  haemoglobin,  contains  less  oxygen,  and  is 
formed  by  reduction  of  the  two  previous. 

4.  Carbon-monoxide  haemoglobin. 

5.  Prussic  acid  methaemoglobin. 

In  examining  the  urine,  especially  the  first  three  vari- 
eties of  haemoglobin  come  into  consideration;  we  give, 
therefore,  in  the  above  table  a  summary  of  their  physical 
and  chemical  properties. 

All  varieties  of  haemoglobin  are  albuminoid  substances, 
and,  therefore,  when  they  are  present,  the  urine  gives  the 
various  reactions  for  albumin. 

The  urine  may  contain  the  various  constituents  of  the 
blood,  haemoglobin,  red  blood-corpuscles,  fibrin  (haema- 
turia) ,  or  only  the  pigment  (haemoglobinuria) .  The  pres- 
ence of  red  blood-corpuscles  and  fibrin  is  detected  micro- 
scopically ;  that  of  pigment  by  the  following  reactions : 

1.  Heller's  Test. — This  reaction  depends  upon  the  for- 
mation of  hsematin  by  the  action  of  sodium  hydrate.  The 
haematin  is  taken  up  by  the  earthy  phosphates  simultane- 
ously with  their  precipitation.  Ten  to  fifteen  cc  of  urine 
are  rendered  strongly  alkaline  with  sodium  hydrate,  and 
heated  to  the  boiling-point;  a  flaky,  red  precipitate  is  pro- 
duced. In  the  presence  of  a  small  quantity  of  haemoglobin 
the  color  becomes  distinct  only  after  the  precipitate  has 
settled. 

If  no  precipitate  is  formed  on  heating  (due  to  the  ab- 
sence of  earthy  phosphates) ,  the  urine  is  treated  with  an 
equal  quantity  of  normal  urine  and  the  test  repeated. 

Following  the  use  of  rheum,  senna,  cascara  sagrada, 
and  santonin,  the  urine  gives  a  similar  reaction.  Urine 
containing  haemoglobin  is  distinguished,  however,  by  the 
fact  that  on  the  careful  addition  of  acetic  acid  only  a  por- 
tion of  the  precipitate  is  dissolved — namely,  the  phos- 
phates— while  the  haematin  remains  in  reddish-brown 


172    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

flakes.  If  the  positive  reaction  was  due  to  drugs,  the  sedi- 
ment and  the  coloration  disappear  absolutely  upon  the 
addition  of  the  acid. 

2.  Almen's  Test  depends  upon  the  transportation  of 
oxygen  from  turpentine  to  guaiacum  resin  by  haemoglobin, 
and  the  consequent  oxidation  of  the  guaiacum. 

Ten  to  fifteen  cc  of  urine  are  covered  with  an  emulsion 
of  equal  parts  of  tincture  of  guaiacum  and  old  (ozonized) 
turpentine.  At  the  point  of  contact  of  the  solutions  a 
ring  is  formed  (due  to  the  precipitation  of  resin)  which 
is  at  first  white,  but,  in  the  presence  of  haemoglobin,  soon 
assumes  a  beautiful  blue  color.  The  test  is;  to  be  sure, 
more  delicate  than  Heller's,  but  it  is  little  suited  for  the 
detection  of  haemoglobin  in  the  urine,  since  animal  cells, 
especially  pus-corpuscles,  may  cause  a  similar  reaction. 

8.  The  Benzidin  Test  is  executed  in  the  same  way,  as 
in  the  examination  of  the  stomach  contents. 

4.  Spectroscopical  Examination. 

Principle. — Each  variety  of  haemoglobin  possess  the 
property  of  absorbing  certain  rays  of  light,  so  that  dark 
stripes  are  formed  in  the  spectrum  (absorption  bands) , 
which  are  characteristic  of  that  variety.  The  best  and 
most  convenient  spectroscopes  for  examining  the  urine  are 
Browning's  or  VogeVs  pocket  spectroscopes.  They  show 
the  absorption  phenomena  even  more  distinctly  and  sharply 
than  the  majority  of  the  larger  forms  of  apparatus.  The 
determination  of  the  position  of  the  absorption  bands  in 
the  spectrum  is  made  in  these  forms  of  apparatus  by  com- 
parison with  the  solar  spectrum,  which  is  simultaneously 
shown  by  means  of  a  special  arrangement  (comparison 
prism).  For  spectroscopical  examination  the  urine  is 
filtered,  and  diluted  as  necessary;  alkaline  urine  is  acidified 
with  acetic  acid.  The  urine  is  then  poured  into  a  recep- 
tacle having  two  parallel  sides  of  colorless  glass  (haema- 


URINE  173 

tinometer)  ; 1  this  is  held  against  the  opening  of  the  spec- 
troscope so  that  the  rays  of  light  (from  a  gas  or  oil  flame 
or  daylight)  pass  through  the  urine  perpendicularly  to  the 
sides  of  the  glass.  In  observing  the  spectrum  the  position 
of  the  absorption  bands  is  determined  by  comparison  with 
the  solar  spectrum,  which,  by  means  of  a  simple  arrange- 
ment, may  be  thrown  in  and  out  of  focus.  The  charac- 
teristic spectra  of  the  varieties  of  haemoglobin  which  come 
into  consideration  in  the  examination  of  urine  can  be  seen 
in  the  table. 

E.     H^MATOPORPHYRIN 

Hsematoporphyrin  may  be  made  from  ha3matin  artifi- 
cially, by  the  action  of  concentrated  sulphuric  acid,  and 
the  treatment  of  the  solution  with  acid  alcohol  and  stan- 
num,  or  zinc.  Ha3matoporphyrin  differs  from  haematin 
only  in  that  it  contains  no  iron.  Hsematoporphyrin  has 
been  detected  in  the  urine  in  various  diseases.  It  also 
very  frequently  appears  following  the  use  of  large  quanti- 
ties of  sulphonal  and  trional.  Urine  containing  hsemato- 
porphyrin  is  brownish-red,  in  thin  layers  yellowish-red. 

Detection. — Twenty  to  twenty-five  cc  of  urine  are  pre- 
cipitated with  a  mixture  of  equal  parts  of  a  saturated 
solution  of  barium  hydrate  and  of  a  10  per  cent,  solution 
of  barium  chloride,  the  precipitate  collected  upon  a  filter, 
and  washed  with  water,  and  once  with  alcohol.  The  pre- 
cipitate is  then  rubbed  with  a  few  drops  of  hydrochloric 
acid  and  a  small  quantity  of  alcohol,  allowed  to  stand 
awhile,  then  heated  on  a  water-bath  and  filtered.  The 
acid,  red  filtrate  shows,  on  spectroscopical  examination, 
two  absorption  bands :  the  first  in  front  of  D,  the  second, 
broader  band,  between  D  and  E. 

1  An  ordinary  test-tube  may  be  used  instead  of  the  hsematin- 
ometer. 


174    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

If  the  solution  is  rendered  alkaline  with  ammonia,  it 
assumes  a  yellow  color,  and  shows,  spectroscopically,  four 
bands  from  red  to  violet.  The  first  and  third  bands  are 
narrow,  the  second  and  fourth  wide. 

F.  MELANIN 

Normal  urine  contains  no  melanin.  Melanuria  is  a 
pathological  phenomenon,  and  appears  in  patients  having 
melanotic  tumors.  Freshly  passed  urine  contains  prob- 
ably only  the  chromogen,  melanogen,  which  is  later  con- 
verted, by  oxidation,  into  melanin.  Urine  containing 
melanin  is  dark  in  color,  and  on  standing  exposed  to  the 
air  becomes  dark  brown  to  black. 

Detection. — If  a  solution  of  ferric  chloride  or  potassium 
bichromate  is  added  to  the  urine  acidified  with  dilute  sul- 
phuric acid,  a  dark  coloration  is  produced.  The  same 
coloration  is  produced  by  the  addition  of  chlorine  or  bro- 
mine water  to  such  acidified  urine.  An  excess  of  the  oxi- 
dizing agent  decolorizes  the  urine  with  the  production  of 
a  dirty  yellow  precipitate. 

14.  Diazo  Reaction 

The  substances  which  cause  this  reaction,  which  was 
suggested  by  Ehrlicli,  are  as  yet  unknown.  Normal  urine 
does  not  give  the  reaction ;  it  appears  only  in  the  urine  in 
febrile  diseases,  most  often  in  typhoid  fever,  tuberculosis, 
and  measles. 

For  the  performance  of  the  test  two  solutions  are 
necessary : 

1.  Sodium  nitrite 0.5 

AquaB  dest 100.0 

2.  Acidum  sulphanilicum  .       .       .         5.0 
Acidum  hydrochloricum        .       .       50.0 
Aqua?  dest 1,000.0 


URINE  175 

Two  cc  of  the  first  and  98  cc  of  the  second  solution  are 
mixed.  The  reaction  is  carried  out  in  the  following 
manner : 

Ten  to  fifteen  cc  of  urine  are  treated  in  a  test-tube 
with  an  equal  quantity  of  the  reagent,  shaken  vigorously 
until  a  foam  is  produced,  and  then  about  1  cc  of  ammonia 
is  added.  The  reaction  is  positive  if  the  foam  and  liquid 
are  both  colored  brilliant  red.  Normal  urine  is  only  colored 
yellow  with  this  test.  After  twenty- four  hours'  standing, 
a  positive  test  throws  down  a  precipitate,  the  upper  por- 
tion of  which  is  blue,  green,  or  black. 

The  urine  gives  a  similar  reaction  following  the  internal 
use  of  naphthaline.  While  following  the  use  of  prepara- 
tions of  tannic  acid  the  previously  pronounced  reaction 
disappears  entirely. 

15.  Adventitious  Constituents  of  the  Urine 

Of  the  great  number  of  adventitious  constituents  of  the 
urine,  the  majority  of  which  follow  the  ingestion  of  drugs, 
only  those  will  be  considered  here  which  in  the  first  place 
are  easy  to  detect,  and  in  the  second  have  a  certain  clini- 
cal or  therapeutic  significance. 

1.  Mercury  (detection  according  to  Stukoivenkoff ) . — 
Five  cc  of  egg-albumin  are  thoroughly  rubbed  in  a  mortar 
with  an  equal  quantity  of  a  saturated  solution  of  sodium 
chloride,  and  dissolved  in  500  cc  of  urine.  The  solution 
is  then  warmed  on  a  water-bath  until  the  albumin  is  com- 
)letely  coagulated.  The  precipitate  is  collected  on  a  fil- 
;er,  dried  between  filter-paper,  and  then  rubbed  in  a  mor- 
iar  with  about  10  cc  of  concentrated  hydrochloric  acid. 
Forty  cc  of  hydrochloric  acid  are  then  added,  and  the  solu- 
iion,  in  which  a  copper  or  brass  spiral  is  placed,  is  allowed 
D  stand  twenty-four  hours  in  a  glass  beaker  or  cylinder. 
The  albumin  and  the  mercury  which  has  been  collected  by 


176    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

it  are  dissolved  by  the  hydrochloric  acid.  The  mercury 
forms  an  amalgam  on  the  surface  of  the  copper  spiral. 
The  spiral  is  washed,  first  with  cold  then  with  hot  water, 
rinsed  in  alcohol  and  ether,  and  dried  in  the  air.  It  is 
placed  in  a  dry  piece  of  narrow  glass  tubing,  which  has 
been  sealed  at  one  end  by  melting.  A  crystal  of  iodine 
is  then  sublimed,  by  slight  heating,  at  the  upper  end  of 
the  spiral.  The  tube  is  carefully  heated  with  continuous 
turning  from  the  lower  to  the  upper  end  of  the  copper 
spiral.  The  mercury  is  thus  sublimed,  and,  combining 
with  the  iodine,  forms  a  brick-red  ring  of  mercuric  iodide. 
The  width  of  the  ring  is,  if  the  instructions  are  carried 
out  exactly,  proportional  to  the  quantity  of  mercury,  and 
a  quantitative  estimation  is  thus  rendered  possible.  It  is 
only  necessary  to  have  a  scale — that  is,  a  series  of  mercuric 
iodide  rings  obtained  from  definite  quantities  of  mercury 
(1,  2,  8,  4,  etc.,  milligrammes) — and  to  compare  the  ring 
obtained  with  this  scale. 

This  method  is  very  delicate ;  0. 0005  gramme  of  mercury 
can  be  clearly  detected.  If  the  ring  is  not  clearly  seen  mac- 
roscopically,  the  characteristic  red  crystals  of  mercuric  io- 
dide can  be  easily  detected  microscopically  with  low  power. 

2.  Arsenic  in  the  urine  is  detected,  according  to  Gutzeit, 
in  the  following  manner :  One  cc  of  urine  is  treated  in  a 
glass  cylinder  or  wide  test-tube  with  4  cc  of  dilute  sul- 
phuric or  hydrochloric  acid,  and  a  piece  of  arsenic-free 
zinc.  The  receptacle  is  closed  with  a  cotton  plug,  and 
covered  with  filter-paper  moistened  with  a  concentrated 
solution  of  silver  nitrate.  The  filter-paper  assumes  a 
lemon-yellow  color,  which  on  longer  standing  turns  black, 
due  to  the  formation  of  metallic  silver  (from  the  yellow 
arsenate  of  silver) .  In  the  urine  this  test  is  quite  reliable, 
as  the  urine  very  rarely  contains  substances  which  can  in- 
fluence the  reaction. 


URINE  177 

3.  Potassium  Iodide  and  Organic  Preparations  of  Iodine 

(iodol,  iodoform,  etc.). — Ten  to  fifteen  cc  of  urine  are 
treated  with  5  to  10  drops  of  yellow  nitric  acid.  One  to 
two  cc  of  chloroform  are  added,  the  test-tube  closed  with 
a  cork,  and  repeatedly  inverted.  The  chloroform  turns  a 
beautiful  violet- red  from  the  liberated  iodine.  The  colora- 
tion disappears  upon  the  addition  of  a  small  quantity  of 
sodium  thiosulphate.  As  has  already  been  mentioned, 
iodoform  separates  out  during  the  indican  test,  and  causes 
a  violet-red  coloration  of  the  chloroform.  This  test  detects 
with  certainty  small  quantities  of  iodine  in  the  urine 
(0.005). 

4.  Potassium  bromide  and  preparations  of  bromine  are 
also  detected  by  the  indican  test.     The  test  is  not  delicate 
(less  than  0. 1  cannot  be  detected) . 

5.  Chrysophanic   acid  (dioxymethylanthrachinon)   ap- 
pears in  the  urine  following  the  use  of  rheum,   senna, 
chrysarobin,  and  cascara  sagrada.      The   urine    has    an 
intense  yellow  or  greenish-yellow  color.     Alkaline  urine  is 
red.     Upon  the  addition  of  alkalies,  the  acid  yellow  or 
greenish-yellow  urine  also  becomes  red.      The  red  color 
disappears  upon  the  addition  of  acetic  acid  (in  contradis- 
tinction to  blood-pigment) .     With  the  indican  test,  chlo- 
roform assumes  a  greenish  coloration. 

Urine  containing  chrysophanic  acid  strongly  reduces 
Nylander**  reagent. 

6.  Salicylic  Acid  and  its  Preparations  (salol,  salipyrin, 
salophen,  etc.). — The  salicylates  appear  in  the  urine  as 
salicyluric  acid,   mono-ethyl-sulphuric  esters,  combined 
with  glycuronic  acid,  and  partially  unaltered,  and  can  be 
easily  detected  a  very  short  time  following  their  ingestion. 
The  urine  has  usually  a  dark  color,  which  deepens  on 
standing. 

For  the  detection  of  salicylic  acid  preparations   the 


178    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

urine  is  treated  with  5  to  10  drops  of  ferric  chloride.  The 
solution  becomes  intensely  blue-violet  in  color;  in  the 
presence  of  smaller  quantities  it  becomes  dark  red.  Since 
other  adventitious  constituents  of  the  urine  (antipyrin, 
phenacetin)  give  a  similar  reaction,  Marcuse  recommends 
the  following  procedure  for  the  identification  of  salicylic 
acid :  The  urine  treated  as  above  is  further  treated  with 
hydrochloric  acid,  a  drop  at  a  time,  until  a  red  color  is 
just  distinctly  present  (upon  the  further  addition,  the  color 
disappears  completely,  owing  to  the  decomposition  of  the 
ferric  salicylate) .  The  solution  is  then  shaken  with  acetic 
ether,  whereupon  the  red  coloration  disappears,  if  due  to 
salicylic  acid;  if  due  to  derivatives  of  antipyrin  or  phe- 
nacetin, the  solution  is  not  decolorized. 

7.  Antipyrin. — Following  the  use  of  large  quantities  of 
antipyrin,  the  urine  is  colored  yellow  to  blood-red,  and 
shows  a  greenish-red  fluorescence. 

Detection. — (a)  A  dark  red  color  is  produced  by  ferric 
chloride,  which  does  not  disappear  upon  boiling  nor  upon 
shaking  with  acetic  ether. 

(b)  If  the  urine  is  treated  with  a  drop  of  acetic  acid 
and  Lugol's  iodine  potassium  iodide  solution,  a  ruby-red 
crystalline  precipitate  is  formed  (Marcuse) . 

8.  Phenacetin  is  excreted  in  the  urine  partly  as  phe- 
netidin,  partly  as  para-am idophenol,  and  partly  in  coupled 
combination  with  glycuronic  acid. 

Detection.  —  (a)  The  urine  is  treated  with  2  drops  of 
hydrochloric  acid  and  2  drops  of  a  1  per  cent,  solution 
of  sodium  nitrate.  If  an  alkaline  watery  solution  of  a- 
naphthol  and  a  little  sodium  hydrate  are  added,  a  red 
coloration  appears,  which,  upon  the  addition  of  hydro- 
chloric acid,  turns  violet. 

(b)  With  ferric  chloride  the  urine  becomes  brownish- 
red. 


URINE  179 

9.  Balsam  of  Copaiba. —  (ft)   Treated  with  hydrochloric 
acid  the  urine  assumes  a  pinkish-red  color,  which  on  boil- 
ing turns  red-violet. 

(b)  In  performing  the  albumin  test  a  heavy  clouding 
is  produced,  which  disappears  upon  the  addition  of  al- 
cohol or  petroleum-ether. 

10.  Urotropin  enters  the  urine  quickly,   and  may  be 
detected  in  it  even  within  half  an  hour  after  its  ingestion 
by  means  of  a  saturated  solution  of  bromine  in  water,  in 
which  it  produces  a  yellow  precipitate,  soluble  in  an  excess 
of   urine.     It   has   not   yet   been   proved  that  urotropin 
liberates    formaldehyde  in  the   urine.     The  latter  is  de- 
tected in  the  urine  by  means  of  phloroglucin  and  sodium 
hydrate.     A  red  color  is  produced. 

11.  Phenol  (carbolic  acid)  is  excreted  in  the  urine  as 
phenol  sulphuric  acid.     The  urine  is  greenish-brown  in 
color,  and  becomes  darker  on  standing.     The  dark  color 
of  carbolic  acid  urine  depends,  according  to  Baumann, 
upon  the  formation  of    hydrochinon.      The  latter,  upon 
further  oxidation,    forms    brown    substances   (not    more 
definitely  known) . 

Detection. — Phenol  cannot  be  detected  directly  in  the 
urine  (since  the  latter  contains  no  free  phenol),  but  must 
first  be  isolated.  As,  however,  normal  urine  contains  a 
small  quantity  of  phenol  compounds  (about  0.03  in  twenty- 
four  hours),  only  a  marked  increase  is  indicative  of  car- 
bolic acid  poisoning.  To  isolate  phenol,  a  la-rge  quantity 
of  urine  is  distilled  after  the  addition  of  sulphuric  acid 
(about  5  to  10  cc  of  sulphuric  acid  to  100  cc  of  urine) 
until  the  phenol,  liberated  from  the  sulphuric  acid  esters, 
is  all  distilled.1  The  distillate  is  neutralized  with  pure 


1  This  is  recognized  by  the  fact  that  the  distillate  no  longer 
clouds,  or  produces  a  precipitate  in  bromine- water. 


180    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

sodium  carbonate  and  redistilled.  This  distillate  gives 
the  following  reactions : 

(a)  Upon  the  addition  of  a  few  drops  of  a  neutral 
solution  of  ferric  chloride  a  blue  coloration  appears. 

(#)  With  bromine-water  a  yellowish-white  precipitate 
of  tribromphenolbrom  is  formed.  The  precipitate  is  dis- 
solved by  sodium  hydrate  and  is  reprecipitated  from  the 
alkaline  solution  by  hydrochloric  acid  as  tribromphenol, 
forming  yellow  crystalline  needles. 

(c)  With  nitrous  acid  nitrogen  is  liberated. 

V.  Quantitative  Chemical  Examination  of 
the  Urine 

1.  Estimation  of  Albumin 

(a)  Method  of  Roberts  and  Stolnikoff,  modified  by 
Brandberg. 

Principle. — If  the  urine  contains  0.0033  gramme  of 
albumin  in  100  cc — i.e.,  0.033  per  litre — the  annular 
clouding  with  Hellers  test  appears  only  after  two  to  three 
minutes.  The  method  depends  upon  this  fact.  The  urine 
to  be  examined  is  diluted  with  water  until  with  Heller's 
test  a  ring  is  formed  only  after  two  to  three  minutes. 
From  the  dilution  necessary  the  albumin  contained  in  the 
undiluted  urine  can  easily  be  calculated. 

Procedure. — First  a  dilution  of  1:10  is  made  with  the 
urine  to  be  examined.  Five  cc  of  urine  are  measured  with 
a  pipette,  put  in  a  glass  cylinder,  and  45  cc  of  water  added. 
The  mixture  is  thoroughly  shaken  and  a  portion  used  for 
Heller's  test.  If  after  two  to  three  minutes  no  ring  is 
formed,  the  dilution  is  too  great,  and  a  dilution  of  1 :  5  or 
1 :  3  is  made  from  the  undiluted  urine.  If,  however,  with 
the  dilution  of  1:10  the  ring  appears  at  once,  the  urine 


URINE  181 

must  be  still  further  diluted  by  making  dilutions  of  1 :  80, 
50,  or  100  from  the  1:10  dilution,  until  a  dilution  is  ob- 
tained with  which  a  ring  appears  only  after  two  to  three 
minutes.  With  a  little  practice  in  carrying  out  this 
method,  it  is  easy  to  approximately  estimate  the  necessary 
dilution  from  the  intensity  of  the  first  clouding,  and, 
therefore  the  entire  estimation  may  be  comparatively 
quickly  made.  The  quantity  of  albumin  per  litre  is  cal- 
culated by  multiplying  the  number  0. 088  by  the  number 
of  the  dilution.  For  example :  If  the  ring  appears  only 
after  two  to  three  minutes  with  a  dilution  of  1 :  80  the  un- 
diluted urine  contains  0.088X80  =  0.99  gramme  of  albu- 
min per  litre.  This  method  yields  results  which  are  suffi- 
ciently accurate  for  clinical  purposes.  It  must,  however, 
be  carefully  and  accurately  carried  out.  It  is  especially 
important  that  Heller's  test  be  each  time  carried  out  lege 
artis. 

(b)  Essbach's  Method. 

Principle. — Essbach's  reagent  is  composed  of  a  solu- 
tion of  10  grammes  of  picric  acid  and  20  grammes  of 
citric  acid  in  a  litre  of  water.  The  albumin  from  a  defi- 
nite amount  of  urine  is  precipitated  with  this  reagent,  and 
from  the  height  of  the  precipitated  albumin  the  amount 
of  albumin  contained  in  the  urine  is  calculated,  according 
to  an  empirical  scale. 

Procedure. — EssbacWs  albuminometer  (a  graduated 
tube)  is  filled  with  the  filtered  urine  to  mark  U.  The 
reagent  is  then  added  to  mark  R,  the  tube  closed  with  a 
rubber  stopper,  and,  without  shaking,  inverted  ten  to 
twelve  times.  The  tube  is  stood  vertically  in  a  standard, 
and  after  twenty-four  hours  the  height  of  the  precipitate 
is  read.  The  numbers  show  in  grammes  the  amount  of 
albumin  contained  in  1,000  cc  of  urine.  The  urine  must 
not  contain  more  than  4  per  cent,  of  albumin;  when  it 


182    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

contains  more  albumin  it  must  be  correspondingly  diluted. 
The  method  does  not  give  accurate  nor  reliable  results. 
A  precipitate  is  frequently  formed  in  urine  containing  no 
albumin. 

(c)  Gravimetric  Analysis. — One  hundred  cc  of  filtered 
urine  are  treated  with  1  to  2  drops  of  acetic  acid,  and 
heated  on  a  water-bath  until  the  albumin  has  precipitated 
in  flakes.  The  precipitate  is  collected  upon  a  filter,  which 
has  been  previously  dried  at  110°  C.,  and  weighed,  washed 
with  water,  and  then  with  alcohol  and  ether,  dried  at  110° 
C.,  and  weighed.  The  filter  and  its  contents  are  then 
burned  in  a  platinum  crucible,  reduced  to  ashes,  weighed, 
and  the  weight  of  the  ashes  subtracted  from  the  weight  of 
the  albumin. 

Instead  of  gravimetric  analysis  the  coagulated  and 
washed  albumin  can  be  treated  according  to  Kjeldahl  (cf. 
p.  187),  and  its  nitrogen  estimated.  The  amount  of 
albumin  is  calculated  by  multiplying  the  amount  of  nitro- 
gen obtained  by  6.25. 

This  method  yields  the  only  accurate  result,  but  is, 
unfortunately,  too  complicated,  and  consumes  too  much 
time  for  clinical  and  practical  use. 

2.  Estimation  of  Sugar 

(a)  By  Polarization. — For  the  quantitative  estimation 
of  sugar  a  Laurent's  apparatus  is  best  used,  which  is 
arranged  for  white  lamplight,  and  allows  the  direct  read- 
ing of  the  percentage  of  sugar  contained.  The  urine  must 
be  especially  prepared  for  polarization — i.e.,  the  following 
requirements  must  be  complied  with : 

1.  The  urine  must  be  absolutely  clear,  and  must  not 
be  deeply  colored.  Turbid  urine  must,  therefore,  be  fil- 
tered, while  highly  colored  urine  must  first  be  decolorized 
with  lead  acetate;  a  few  knife-points  of  powdered  neutral 


URINE  183 

lead  acetate  are  added  to  about  50  cc  of  urine,  the  urine 
thoroughly  shaken  and  filtered  through  a  dry  filter. 

2.  The  urine  must  be  free  from  albumin,  since  albumin 
is  leevorotary.  When  but  a  slight  amount  is  present 
(under  0.1  per  cent.),  this  laevorotation  may  be  ignored; 
when  more  is  present,  the  albumin  must  be  removed  by 
boiling  and  the  urine  brought  up  to  its  original  volume. 

The  clear,  as  nearly  as  possible  colorless,  urine  is 
poured  into  the  observation  tube  of  the  polarimeter,  care 
being  taken  that  it  forms  a  convexity  above  the  end  of  the 
tube;  the  cover-glass,  which  must  be  absolutely  clean  and 
dry,  is  slipped  on  from  the  side,  so  that  no  air  is  included ; 
the  brass  cap  is  then  adjusted.  The  apparatus  is  placed 
at  the  zero-point,  and  the  observation-tube  in  it.  If  the 
urine  contains  sugar,  the  right  half  of  the  field  will  be 
darkened.  The  apparatus  is  adjusted  by  turning  the  screw 
so  that  both  halves  of  the  field  are  equally  bright.  The 
scale  then  shows  the  percentage  of  sugar  present. 

Polarization  is  a  sufficiently  accurate  method  for  prac- 
tical use.  Large  quantities  of  glycuronic  compounds, 
£-oxybutyric  acid,  and  levulose  can,  however,  cause  error. 

(b)  Fermentation  Test  According  to  Roberts. 

Principle. — The  amount  of  sugar  is  estimated  from  the 
difference  in  the  specific  gravity  of  the  urine  before  and 
after  fermentation.  Roberts  determined  by  investigation 
that  a  reduction  of  the  specific  gravity  of  0.001  represents 
0.280  per  cent,  of  sugar. 

Procedure. — The  specific  gravity  at  15°  C.  is  deter- 
mined, and  100  to  200  cc  of  urine  are  fermented  in  a  flask 
with  yeast  (a  piece  the  size  of  a  hazel-nut) .  After  twenty- 
four  to  thirty-six  hours  the  urine  is  tested  (by  the  ordi- 
nary qualitative  tests)  to  see  if  the  sugar  has  entirely  dis- 
appeared. If  this  is  the  case,  the  specific  gravity  at  15° 
C.  is  again  determined.  Example: 


184    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Specific  gravity  before  fermentation     .       .     1.030 
Specific  gravity  after  fermentation       .       .     1.020 

Difference          .       .     0.010 
The  urine  contains  0. 280  X 10  =  2. 80  per  cent,  of  sugar. 

The  method  gives  comparatively  accurate  results  when 
the  determination  of  the  specific  gravity  is  very  carefully 
carried  out,  and  the  urine  contains  nqt  less  than  0.5  per 
cent,  of  sugar. 

(c)  Fermentation  Test  According  to  Lohnstein. — Lohn- 
steirfs  eaccharometer  consists  of  a  U-shaped  tube,  the 
short  arm  of  which  has  a  bulbous  enlargement,  and  can  be 
hermetically  closed  with  a  stopper,  which  has  a  perfora- 
tion corresponding  to  a  second  in  the  neck  of  the  bulb.  A 
scale,  a  Pravaz  syringe,  a  weight,  a  special  grease,  and  a 
bottle  of  mercury  are  supplied  with  the  apparatus. 

Sugar  is  estimated  in  the  following  manner :  The  mer- 
cury is  poured  into  the  apparatus  while  both  ends  of  the 
tube  are  open;  0.5  cc  'of  the  urine  to  be  examined  are 
placed,  with  the  Pravaz  syringe,  upon  the  surface  of  the 
mercury  within  the  bulb.  The  syringe  is  then  washed 
with  ordinary  water,  and  1°  to  2°  (of  the  scale  on  the 
syringe)  of  yeast  paste  (prepared  with  a  piece  of  com- 
pressed yeast  and  a  few  drops  of  water)  are  added.  The 
stopper  is  greased  and  placed  in  the  neck  of  the  bulb,  so 
that  its  perforation  corresponds  to  that  in  the  neck  of  the 
bulb.  The  scale  is  now  set  upon  the  tube  of  the  apparatus. 
If  the  meniscus  of  the  mercury  is  not  exactly  at  the  level 
of  the  zero  line,  it  is  placed  there  by  tipping  the  appara- 
tus, and  the  stopper  is  then  turned  so  that  the  holes  do 
not  correspond  with  one  another.  The  weight  is  placed  on 
the  stopper,  and  the  urine  left  to  ferment  in  the  apparatus, 
either  at  room-temperature  or  in  an  incubator.  At  a  tem- 
perature of  82°  to  88°  C.  fermentation  is  completed  at  the 


URINE  185 

end  of  ^three  to  four  hours,  even  when  considerable  sugar 
is  present;  at  ordinary  room-temperature  it  takes  six  to 
eight  hours.  On  the  removable  frame  are  two  scales,  one 
of  which  is  for  20°  C.,  and  the  other  for  35°  C.  In  the 
majority  of  cases  the  20°  scale  can  be  used  without  marked 
error.  A  more  exact  estimation  can  be  made  by  means  of 
the  following  formula: 


In  which  p20  and  p35  are  the  readings  on  the  two  scales, 
and  T  is  the  temperature  at  which  the  apparatus  was 
filled,  and  to  which  it  has  been  again  cooled  at  the  com- 
pletion of  the  fermentation. 

After  the  estimation  the  apparatus  must  be  cleansed. 
The  stopper  is  first  turned  so  that  the  perforations  corre- 
spond, then  removed,  the  mixture  of  urine  and  yeast 
swabbed  out  with  a  bit  of  cotton,  and  the  bulb  washed  out 
with  a  stream  of  water  until  the  water  runs  away  clear. 
The  rest  of  the  water  is  removed  with  absorbent  cotton. 
Lohnstein's  apparatus  is  to  be  highly  recommended  for 
clinical  and  practical  use.  It  is  especially  serviceable  in 
cases  in  which  the  percentage  of  sugar  is  very  small,  since 
it  allows  accurate  readings  of  0.1  per  cent.,  or  even  0.05 
per  cent. 

(d)  By  f  if  ration  after  Pavy-Salili.  —  Solutions  required  : 

SOLUTION  No.  1 

Cupri  sulfurici  crystallisati  puri     .       .       4.  158 
Aquae  destillatae  ad  ......  500.0 

SOLUTION  No.  2 
Salis  Seignetti      ......     20.4 

Kali  caustici  puri        .....     25.0 

Ammonii  caustici  (sp.  gravity,  0.88)   .  800.0 
Aquae  destillatae  ad  ......  500.0 


186    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Method  of  Determination. — 5  cc  of  each  of  these  solu- 
tions with  30  cc  water  are  put  into  an  Erlenmeyer  flask  of 
75  to  100  cc.  The  flask  is  put  over  a  Bunsen  burner  upon 
an  asbestos  wire  netting  and  we  wait  until  the  contents  in 
the  flask  begins  to  boil.  In  order  better  to  regulate  the 
flame,  a  piece  of  copper  wire  netting  is  placed  over  the 
opening  of  the  Bunsen  burner.  While  the  solution  is  be- 
ing heated,  the  urine  to  be  examined  is  diluted  ten  times 
(5  cc  urine  +  45  cc  of  water)  and  this  diluted  solution  is 
put  into  a  burette.  As  soon  as  the  solution  in  the  flask 
begins  to  boil,  the  flame  is  reduced  somewhat,  so  that  it 
may  continue  to  boil  slowly.  The  burette  is  taken  into 
the  left  hand  and  the  urine  solution  is  allowed  to  flow  into 
the  boiling  flask  from  the  burette,  drop  by  drop,  care 
being  taken  that  the  boiling  be  not  interrupted  until  the 
blue  solution  of  the  flask  is  entirely  decolorized.  We  note 
the  number  of  cubic  centimetres  of  the  urine  solution  used. 
The  determination  is  to  be  considered  as  at  an  end  only 
then,  when  the  number  of  cubic  centimetres  used  up  is 
between  5  and  10.  If  less  than  5  cc  have  been  used  a 
second  determination  must  be  made  whereby  a  greater 
dilution  of  the  urine  is  made. 1  The  quantity  of  the  urine 
dilution  used  up  at  the  first  titration  guides  us  in  making 
the  second  dilution.  If  at  the  first  titration  less  than  1  cc 
was  used  up,  we  prepare  another  solution  of  the  urine 
which  has  been  diluted  one  hundred  times. 

If  1     to  1.5  cc  were  used  then  a  dilution  of  50 
"  1.5  to  2.5  "          "  "  "         40 

"  2.5  to  5.0  "          "  "  "         20 


1  The  second  dilution  is  absolutely  necessary,  as  accurate  results 
can  be  obtained  only  if  the  urine  contains  0.5  to  1.0  sugar  per 
mille. 


URINE  187 

The  sugar  determination  is  made  in  the  following  manner. 
0.005  gr.  of  grape-sugar  is  required  to  reduce  10  cc  of 
Pavy's  solution  used.  Therefore  the  used  up  quantity  of 
the  urine  solution  contains  0. 005  glycose.  If  at  the  second 
titration  8  cc  were  used  of  the  urine  which  was  diluted  20 
times,  then  such  urine  contains 

0.005    .  20  .   100 

— r —       — «•  =  l.zo  per  cent,    sugar, 
o 

The  examination  lasts  ten  to  twenty  minutes. 

3.  Estimation  of  Total  Nitrogen 

The  nitrogen  of  the  urine  is  usually  estimated  accord- 
ing to  KjeldaliVs  method. 

Principle. — The  various  nitrogenous  substances  are 
converted  into  ammonium  sulphate  by  boiling  with  sul- 
phuric acid.  The  ammonia  is  freed  from  the  ammonium 
sulphate  by  supersaturation  with  a  solution  of  sodium 
hydrate,  and  collected  in  a  titrated  solution  of  sulphuric 
acid.  From  the  amount  of  acid  bound  by  the  ammonia, 
the  NH3  contained  is  calculated,  and  from  it  the  N  is  cal- 
culated. 

Procedure. — Five  cc  of  urine  are  treated  in  a  so-called 
Kjeldalil-ft&$k.  (of  hard  glass)  with  10  cc  of  Kjeldalil- 
sulphuric  acid  (a  mixture  of  three  parts  pure  and  one 
part  fuming  sulphuric  acid)  and  a  few  drops  of  a  saturated 
solution  of  copper  sulphate.  The  flask  is  then  heated  on 
a  sand-bath  in  a  fume-chamber  until  the  solution  is 
decolorized.  The  solution  is  then  allowed  to  cool,  and, 
with  agitation,  50  cc  of  distilled  water  are  added.  The 
solution,  which  has  again  become  hot,  is  poured  into  a 
litre  distilling-flask,  and  the  Kjeldahl-&a&b  rinsed  two  or 
three  times  with  water,  which  is  also  poured  into  the  dis- 
tilling-flask. The  solution  is  now  rendered  alkaline  with 


188    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

40  per  cent,  sodium  hydrate  (about  40  to  60  cc)1  and  dis- 
tilled at  once.  The  addition  of  the  sodium  hydrate  must 
be  made  quickly  to  avoid  loss  of  ammonia.  The  distill- 
ing-tube  must  have  a  bulb  to  prevent  the  sodium  hydrate 
from  being  carried  over  into  the  receiver.  The  distillate 
is  collected  in  a  receiver  containing  50  cc  of  decinormal 
sulphuric  acid.  The  distillation  is  completed  twenty  to 
thirty  minutes  after  boiling  has  begun,  which  is  indicated 
by  marked  bumping  (due  to  beginning  precipitation  of 
sodium  sulphate) .  The  stopper  is  then  removed  from  the 
distill  ing- flask,  the  flame  extinguished,  the  distilling-tube 
washed  with  water  into  the  receiver,  and  the  contents  of 
the  latter  titrated  with  decinormal  sodium  hydrate,  using 
rosolic  acid  as  an  indicator.  The  alkali  is  added  until  a 
permanent  pink  coloration  of  the  solution  is  produced. 
The  number  of  cc  of  decinormal  alkali  used  is  subtracted 
from  the  number  of  cc  of  decinormal  sulphuric  acid  used 
in  the  receiver.  The  difference  multiplied  by  0.0014  gives 
the  number  of  grammes  of  nitrogen  contained  in  the  quan- 
tity of  urine  used,  from  which  the  percentage  is  calculated. 
It  is  advisable,  as  a  control,  to  carry  out  simultaneously 
two  tests  with  samples  of  the  same  urine. 

4.  The  Determination  of  Urates 

(a)  Pflueger-Bleibtreu's  Method. 

Principle.— This  method  depends  on  the  principle  that 
with  the  exception  of  the  urates  all  other  nitrogen  com- 
pounds of  the  urine  are  precipitated  by  phospho-tungstic 
acid.  The  urates  are  then  removed  by  phosphoric  acid 
and  the  nitrogen  contained  in  them  is  determined  after 
Kjeldahl. 

1  If  the  sp.  gr.  of  the  urine  is  higher  than  1020,  75  cc.  of  ^  nor- 
mal sulphuric  acid  is  added. 


URINE  18d 

The  necessary  reagents  are : 

1.  A  solution  of  phospho-tungstic  acid  (9  parts  of  a 
10  per  cent,  phospho-tungstic  acid  plus  one  part  hydro- 
chloric acid  of  the  sp.  gr.  124) . 

2.  Phosphoric  acid  crystals. 
8.   Powdered  calcium  hydrate. 

4.  The  reagents  necessary  for  a  Kjeldahl  determina- 
tion. 

Determination:  Having  previously  determined  the 
absolute  quantity  of  phospho-tungstic  acid  required  for 
the  absolute  precipitation,  such  quantity  of  the  acid  is 
added  to  50  cc  of  the  urine  into  a  flask  of  200  cc.  This  is 
diluted  up  to  150  cc  with  a  10  per  cent,  solution  of  HC1; 
filtration  after  twenty-four  hours;  the  clear  filtrate  is 
rubbed  up  with  the  calcium  hydrate  powder  until  the  reac- 
tion is  alkaline;  again  filtration;  15  cc  (corresponding  to 
5  cc  of  urine)  to  which  10  gr.  of  the  crystalline  phosphoric 
acid  was  added,  are  put  into  a  flask,  which  is  then  left  in 
a  drying  oven,  kept  at  a  temperature  of  150°,  for  four  and 
one  half  hours  and  the  day  substance,  left  after  the  evapo- 
ration, is  dissolved  in  warm  water  and  the  nitrogen  is 
determined  after  Kjeldalil.  The  quantity  of  nitrogen 
obtained  is  multiplied  by  ±f  =2{.  In  this  way  are  deter- 
mined the  urates  of  5  cc  of  urine.  There  is  a  difference 
of  opinion  among  authorities,  whether  ammonia  is  pre- 
cipitated by  the  phospho-tungstic  acid,  or  whether  it 
passes  into  the  filtrate  together  with  the  urates. 

Oumlicli  holds  that  all  the  ammonia  is  precipitated  by 
Merctfs  preparation  of  the  phospho-tungstic  acid.  Whilst 
Pflueger  and  Bleibtreu  maintain  that  the  ammonia  is 
determined  together  with  the  urates  by  the  method 
described.  They  therefore  recommend,  that  the  ammonia 
be  determined  after  the  method  of  Scliloesing  and  that  the 
quantity  of  nitrogen  thus  determined  be  deducted  from 


190     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

what  has  been  previously  obtained  by  phospho-tungstic 
acid.  The  nitrogen  of  the  urates  equals  the  difference  of 
these  two  determinations. 

(/>)  Knop  and  Huefner's  Method. 

Principle.  —  The  urea  is  decomposed  with  an  alkaline 
solution  of  sodium  hypobromite  into  nitrogen,  carbon 
dioxide,  and  water. 


CO  <      7  +  SBrONa  =  CO2  +  2H20  +  2N  +  SNaBr. 


The  carbon  dioxide  is  bound  by  the  soda,  while  the 
quantity  of  nitrogen  liberated  is  estimated  volumetrically. 
From  this  the  urea  is  calculated. 

Procedure.  —  The  estimation  is  best  carried  out  with 
the  apparatus  recently  constructed  by  Jolles.  Jolles'  azo- 
tometer  (Fig.  24)  consists  of  a  mixing-jar  and  two  gradu- 
ated tubes.  These  three  parts  of  the  apparatus  are  joined 
by  rubber  tubing.  The  mixing-jar  (c)  contains  a  smaller 
cylindrical  jar  of  hard  rubber  or  glass.  In  addition  to 
the  rubber*  tube  connecting  the  mixing-  jar  with  the  gradu- 
ated tubes,  a  second  tube  with  a  free  end  passes  through 
the  rubber  stopper  of  the  jar.  This  tube  is  closed  at  its 
outer  end  with  a  pinch-cock.  The  urea  is  estimated  in 
the  following  manner: 

Ten  cc  of  the  filtered  urine  are  treated  in  a   100  cc 
graduated  flask  with  about  30  cc  of  distilled  water,  and 
sufficient  phospho-tungstic  acid  containing  hydrochloric  \ 
acid  for  precipitation  (100  cc  of  HC1  of  a  specific  gravity  of 
1.  124  +  900  cc  of  10  per  cent,  phospho-tungstic  acid)  .    The 
flask  is  heated,   with   agitation,   in    a  water-  bath  for  a 
quarter  of  an  hour,  allowed  to  stand  four  hours,  filled  to 
the  mark  with  distilled  water,  shaken,  and  the  contents 
filtered  through  a  dry  filter.     Twenty-five  cc  of  the  filtrate  j 
(  =  2.5  urine)  are  withdrawn  with  a  pipette,  placed  in  the 


URINE 


191 


FIG.  24. 


192  CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

mixing-jar  of  the  azotometer,  and,  with  agitation,  care- 
fully rendered  alkaline.  Thirty  cc  of  bromine  solution 
(100  grammes  of  sodium  hydrate  are  dissolved  in  250  cc 
of  water,  and  25  grammes  of  bromine  are  added  to  the 
cold  solution)  are  placed  in  the  smaller  inner  vessel.  The 
inner  vessel  is  best  held  in  a  pair  of  forceps  when  intro- 
duced, to  avoid  mixing  the  two  fluids.  The  graduated 
tubes  are  now  filled  with  ordinary  water  to  the  mark  0, 
with  the  pinch-cock  on  the  tube  of  the  mixing-jar  open, 
in  such  a  manner  that  the  liquid  in  the  two  tubes  is  at 
the  same  level.  The  pinch-cock  is  then  closed  and  the 
mixing- jar  inclined,  so  that  the  two  fluids  mix,  whereupon 
a  lively  formation  of  gas  takes  place.  The  water  which 
rises  in  the  right-hand  tube  is  allowed  to  escape  through 
the  outlet,  which  is  near  the  bottom  of  the  tube,  until  its 
level  is  about  1  to  2  centimetres  higher  than  that  of  the 
water  in  the  other  tube.  The  jar  is  shaken  a  few  minutes, 
care  being  taken  that  no  liquid  enters  the  free  end  of  the 
glass  tube.  After  gas  formation  has  ceased  at  least  fifteen 
minutes,  the  water  in  the  tubes  is  brought  to  the  same 
level,  and  the  level  noted.  From  the  number  of  cc  of 
nitrogen  developed,  the  urea  is  calculated  in  the  following 
manner : 

The  temperature  and  the  height  of  the  barometer  are 
estimated,1  and  the  number  of  cc  of  N  found  is  multiplied 
by  the  coefficient  in  the  table  on  pp.  194,  195,  correspond- 
ing to  the  temperature  and  pressure. 

Example. — 18.6  cc  of  nitrogen,   temperature  16°  C., 
height  of  barometer  760,  coefficient  in  the  table  0.998. 
The  amount  of   urea    contained  is    0.998X18.6=18.56; 
grammes  per  litre.     Thus  the  table  renders  possible  afi 


1  As  seen  in  Fig.  24,  there  are  added  to  the  apparatus  a  ther- 
mometer (T)  and  a  barometer  (B). 


URINE  193 

direct  estimation  of  the  amount  of  urea  per  litre.  For 
rapid  practical  use  the  estimation  of  urea  with  the  azo- 
tometer  can  be  much  simplified  by  omitting  the  precipi- 
tation with  phospho-tungstic  acid.  The  entire  estimation 
then  takes  but  fifteen  to  twenty  minutes;  2.5  cc  of  urine 
are  measured  with  an  accurate  pipette  and  placed  in  the 
small  jar.  Thirty 'cc  of  the  bromine  solution  and  100  cc 
of  distilled  water  are  placed  in  the  mixing-jar,  and  the 
nitrogen  estimated  in  the  above-described  manner.  The 
urea  is  calculated  from  the  table  on  pp.  194  and  195. 

5.  Estimation  of  Uric  Acid 

(a)  Hopkins' Method. — One  hundred  cc  of  urine  are  satu- 
rated with  25  grammes  of  sodium  chloride,  and  set  aside 
for  twenty-four  hours.  The  precipitate  is  then  collected 
on  a  filter  and  washed  free  from  chlorine  with  a  10  per 
cent,  solution  of  ammonium  sulphate — i.e.,  until  the  fil- 
trate no  longer  becomes  clouded  upon  the  addition  of  a 
solution  of  silver  nitrate.  The  precipitate  is  then  washed, 
without  loss,  with  hot  water  into  an  Erlenmeyer  flask,  and 
the  solution  allowed  to  cool.  Twenty  cc  of  concentrated 
sulphuric  acid  are  now  added,  and  the  solution,  which 
has  again  become  hot,  is  at  once  titrated  with  a  -^5-  normal 
permanganate  solution.  The  permanganate  solution  is 
added  until  the  red  coloration,  which  is  produced,  lasts  a 
few  seconds.  Each  cc  of  the  permanganate  solution  rep- 
resents 0.00861  gramme  of  uric  acid. 

This  method  gives  comparatively  accurate  and  useful 
results. 

(/')  Kowarsky's  Simplified  Method. 

Exactly  10  cc  of  urine  are  measured  off  with  a  pipette 
and  put  into  a  thin  walled  centrifuge  tube  of  about  15  cc. 
Two  to  three  drops  ammonia  and  3  gr.  of  powdered  am- 
monium chlorid  are  added.  (The  ammonium  chlorid 


194    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 


TABLE   FOR  THE 
1  cc  of  nitrogen  represents  grammes  of 


Height  of 
Barometer. 

10° 

12° 

14° 

15° 

16° 

700  

0.934 

0.943 

0.926 

0.922 

0.917 

702  

0.945 

0.937 

0.929 

0.925 

0.920 

704   ... 

0.948 

0.940 

0.932 

0.927 

0.923 

706  

0.951 

0.943 

0.934 

0.930 

0.926 

'708  

0.954 

0.945 

0.937 

0.932 

0.928 

710  .. 

0.957 

0.948 

0.939' 

0.935 

0.931 

712  

0.959 

0.951 

0.942 

0.938 

0.933 

714  

0.962 

0.953 

0.945 

0.941 

0.936 

716  

0.964 

0.956 

0.948 

0.944 

0.938 

718  

0.967 

0.959 

0.951 

0.946 

0.941 

720  

0.970 

0.962 

0.953 

0.949 

0.944 

722  

0.973 

0.964 

0.956 

0.951 

0.947 

724  ... 

0.975 

0.967 

0.958 

0.954 

0.950 

726 

0.978 

0.970 

0.961 

0.957 

0.952 

728  
730  

0.981 
0.984 

0.973 
0.975 

0.964 
0.967 

0.959 
0.962 

0.955 
0.957 

732  ...  . 

0.987 

0.978 

0.969 

0.965 

0.960 

.734  

0.989 

0.981 

0.972 

0.968 

0.963 

736  

0.992 

0.983 

0.975 

0.970 

0.966 

738  

0.995 

0.986 

0.977 

0.973 

0.969 

740   

0.998 

0.989 

0.980 

0.975 

0.971 

742  
744  

1.000 
1.003 

0.992 
0.994 

0.982 
0.985 

0.978 
0.981 

0.974 
0.976 

746  

1.005 

0.997 

0.988 

0.983 

0.979 

748  

1.008 

0.999 

0.991 

0.986 

0.981 

750  

1.011 

1.002 

0.993 

0.989 

0.984 

752  

1.014 

1.005 

0.996 

0.992 

0.987 

754  

1.017 

1.008 

0.999 

0.994 

0.990 

756  

1.019 

1.011 

1.001 

0.997 

0.992 

758  

1.022 

1.013 

1.004 

0.999 

0.995 

760  

1.025 

1.016 

1.007 

1.002 

0.998 

762 

1.028 

1.018 

1.010 

1.005 

1.000 

764 

1.030 

1.021 

1.012 

1.008 

1.003 

766  

768 

1.033 
1.036 

1.024 
1.027 

1.015 
1.017 

1.011 
1.013 

1.006 
1.008 

770.. 

1.039 

1.029 

1.020 

1.016 

1.011 

URINE 


195 


ESTIMATION   OF   UREA. 

urea  per  litre  ;  temperature  in  Centigrade. 


17° 

18° 

19° 

20° 

21° 

23° 

25° 

0.913 

0.909 

0.904 

0.900 

0.895 

0.886 

0.877 

0.916 

0.911 

0.907 

0.903 

0.898 

0.889 

0.879 

0.919 

0.914 

0.909 

0.905 

0.901 

0.891 

0.882 

0.921 

0.917 

0.912 

0.908 

0.903 

0.894 

0.885 

0.924 

0.920 

0.915 

0.910 

0.906 

0.897 

0.887 

0.927 

0.922 

0.917 

0.913 

0.909 

0.899 

0.890 

0.929 

0.925 

0.920 

0.916 

0.911 

0.902 

0.892 

0.932 

0.927 

0.923 

0.919 

0.914 

0.904 

0.895 

0.934 

0.930 

0.926 

0.921 

0.916 

0.907 

0.897 

0.937 

0.933 

0.928 

0.924 

0.919 

0.910 

0.900 

0.940 

0.935 

0.931 

0.927 

0.921 

0.912 

0.903 

0.943 

0.938 

0.933 

0.929 

0.924 

0.915 

0.905 

0.945 

0.940 

0.936 

0.932 

0.927 

0.917 

0.908 

0.948 

0.943 

0.938 

0.934 

0.930 

0.920 

0.910 

0.951 

0.946 

0.941 

0.937 

0.933 

0.922 

0.913 

0.954 

0.949 

0.944 

0.939 

0.935 

0.925 

0.915 

0.956 

0.951 

0.947 

0.942 

0.938 

0.928 

0.918 

0.959 

0.954 

0.950 

0.945 

0.940 

0.931 

0.921 

0.961 

0.957 

0.952 

0.947 

0.943 

0.933 

0.923 

0.964 

0.959 

0.955 

0.950 

0.945 

0.936 

0.926 

0.967 

0.962 

0.957 

0.952 

0.948 

0.938 

0.928 

0.969 

0.964 

0.960 

0.955 

0.951 

0.941 

0.931 

0.972 

0.967 

0.962 

0.958 

0.953 

0.944 

0.934 

0.975 

0.970 

0.965 

0.961 

0.956 

0.946 

0.937 

0.977 

0.973 

0.968 

0.963 

0.958 

0.949 

0.939 

0.980 

0.975 

0.970 

0.966 

0.961 

0.951 

0.942 

0.982 

0.978 

0.973 

0.968 

0.963 

0.954 

0.945 

0.985 

0.981 

0.975 

0.971 

0.966 

0.957 

0.947 

0.988 

0.983 

0.978 

0.974 

0.969 

0.959 

0.950 

0.991 

0.986 

0.981 

0.976 

0.971 

0.962 

0.952 

0.993 

0.988 

0.984 

0.979 

0.974 

0.964 

0.955 

0.996 

0.991 

0.987 

0.981 

0.976 

0.967 

0.957 

0.999 

0.993 

0.989 

0.984 

0.979 

0.969 

0.960 

1.001 

0.996 

0.992 

0.987 

0.981 

0.972 

0.963 

1.004 

0.999 

0.994 

0.989 

0.984 

0.974 

0.965 

1.006 

1.002 

0.997 

0.992 

0.987 

0.977 

0.968 

196    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

powder  may  be  ordered  in  doses  of  8  gr.  from  the  druggist. ) 
The  tube  is  closed  up  with  a  rubber  stopper  and  thoroughly 
shaken  up  until  all  of  the  ammonium  chloride  is  dissolved. 
Ammonium  urate  is  separated  in  the  form  of  a  flocculent 
sediment.  The  phosphates,  which  are  likewise  sedimented, 
do  not  interfere  with  the  determination.  The  tube  is  left 
for  two  hours  so  that  all  the  ammonium  muriate  may 
separate.  The  tube  is  then  centrifugalized  for  one  to  two 
minutes  which  causes  all  of  the  sediment  to  settle  at  the 
bottom  of  the  tube ;  the  clear  fluid  is  then  poured  off,  usu- 
ally without  any  loss  of  the  sediment.  While  pouring  off 
the  fluid  the  tube  should  be  inclined  but  once,  as  by  re- 
peated inclinations  the  sediment  is  disturbed,  whereby 
some  of  it  may  be  lost. 

The  sediment,  to  which  five  drops  of  concentrated  HC1 
is  added,  is  carefully  heated  over  a  small  flame;  the  am- 
monium muriate  is  thereby  dissolved  and  the  separation 
of  the  free  uric  acid  begins  at  once  in  the  form  of  a  crystal- 
line sediment.  The  tube  is  left  alone  for  one  hour  for  the 
thorough  separation  of  the  uric  acid.  The  separated  uric 
acid  is  shaken  up,  about  2  cc  of  water  are  added  and  then 
centrifugalized;  about  ten  revolutions  are  sufficient  to 
throw  down  the  crystalline  sediment  to  the  bottom;  the 
fluid  is  now  poured  off,  the  sediment  again  shaken  up 
with  2  to  3  cc  of  alcohol  and  again  centrifugalized.  Two  or 
three  more  times  the  sediment  is  washed  with  alcohol  until 
the  alcohol  reacts  neutral  to  litmus.  The  washing  of  the 
sediment  lasts  mostly  from  three  to  five  minutes.  After 
the  last  alcohol  was  poured  off  a  few  cc  of  water  are  heated 
in  a  test-tube ;  about  two  cc  of  the  hot  water  is  poured  over 
the  again  shaken  up  sediment,  one  drop  of  phenol-phtha- 
lein  is  added  and  the  hot  solution  is  titrated  with  a  -fa  nor- 
mal solution  of  piperidin.  This  latter  solution  is  added 
to  the  hot  solution  so  long  (the  hot  solution  being  shaken 


URINE  197 

all  the  time  while  the  titration  lasts)  until  this  hot  solu- 
tion remains  of  a  pink  color  permanently,  even  after  it  is 
heated  up  again. 

To  get  the  number  of  mg  of  uric  acid  contained  in  10 
cc  of  urine,  we  multiply  the  number  of  cc  of  the  piper- 
idin  solution  used  up  in  the  titration  by  8.86.  If,  for 
instance,  1.5  cc  of  the  piperidin  solution  have  been  used 
up  in  the  titration,  we  find  the  number  of  mg  of  uric 
acid  contained  in  10  cc  of  urine  by  multiply  ing  3.86x1.5 
=  5.04  mg.  Therefore  in  100  cc  5.04x10  =  50.4  mg  or 
0.0504  g.,  i.e.,  0.0504  per  cent.  The  piperidin  solu- 
tion can  be  kept  for  some  time  and  its  usability  can 
be  tested  with  a  fa  normal  HC1  or  sulphuric  acid  solu- 
tion. 

(c)  Salkowski  and  Ludwig's  Method. 

Principle. — The  uric  acid  is  precipitated  as  a  salt  of 
silver  and  magnesium,  and  the  uric  acid  liberated  from 
the  silver  precipitate  is  determined  gravimetrically,  or 
calculated  from  the  nitrogen  estimated  according  to 
Kjeldahl. 

NECESSARY  SOLUTIONS. — 1.  An  Ammoniacal  Solution 
of  Silver. — Twenty-six  grammes  of  silver  nitrate  are  dis- 
solved in  water  in  a  litre  flask,  ammonia  is  added  until 
the  precipitate  formed  is  redissolved,  and  water  added  to 
the  mark. 

2.  Magnesium  Mixture. — One  hundred  grammes  of 
crystalline  magnesium  chloride  are  dissolved  in  water  in 
a  litre  flask,  ammonia  added  until  the  mixture  smells 
strongly  of  it,  and  then  a  cold  saturated  solution  of  am- 
monium chloride  added,  until  the  precipitate  of  magne- 
sium hydrate  which  is  formed  is  redissolved;  the  flask  is 
then  filled  to  the  mark  with  water. 

8.  A  Solution  of  Potassium  (or  Sodium)  Sulphate. — 
Fifteen  grammes  of  potassium  hydrate  or  10  grammes  of 


198     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

sodium  hydrate  are  dissolved  in  a  litre  of  water ;  half  the 
solution  is  saturated  with  hydrogen  sulphide,  and  then 
mixed  with  the  other  half. 

Procedure. — Ten  cc  of  magnesium  mixture  and  10  cc 
of  the  silver  solution  are  mixed  in  a  beaker,  and  treated 
with  ammonia  until  the  precipitate  of  silver  chloride  is 
redissolved.  One  hundred  cc  of  urine  are  added  to  the 
mixture,  with  stirring.  The  precipitate  containing  uric 
acid  which  is  formed  at  once  is  collected  on  a  filter,  but  it 
is  not  necessary  to  remove  the  precipitate  which  has  col- 
lected on  the  sides  and  bottom  of  the  beaker.  The  filter 
and  the  precipitate  are  placed  in  the  same  beaker  in  which 
the  precipitation  was  carried  out.  Ten  cc  of  the  potassium 
sulphate  solution  and  10  cc  of  water  are  added  and  heated 
to  the  boiling-point  (prolonged  and  vigorous  heating  is 
to  be  avoided,  as  the  uric  acid  can  be  oxidized  by  it) . 
The  hot  solution  is  filtered,  and  the  residue  washed  with 
hot  water.  The  filtrate  is  collected  in  a  porcelain  dish, 
and  the  sodium  urate  contained  in  it  decomposed  by  the 
addition  of  a  small  amount  of  hydrochloric  acid.  After 
it  has  evaporated  down  to  about  15  cc,  and  a  few  more 
drops  of  hydrochloric  acid  have  been  added,  it  is  set  aside 
for  twelve  to  twenty- four  hours.  The  precipitated  free 
uric  acid  is  collected  on  a  small  weighed  filter,  washed 
with  water,  ether,  alcohol,  and  carbon  bisulphide,  dried, 
and  weighed.  The  amount  of  the  crystallized  uric  acid 
can  be  more  simply  determined  by  the  estimation,  ac- 
cording to  Kjeldalil,  of  the  nitrogen  contained.  The 
uric  acid  collected  on  the  filter  is  then  washed  with  a 
small  quantity  of  water,  the  filter  and  precipitate  placed 
in  a  A}W#/£/-flask,  and  the  further  estimation  carried 
out  as  in  estimating  nitrogen  in  the  urine.  The  quan- 
tity of  nitrogen  contained  is  multiplied  by  3.  This 
method  is  more  complicated,  and  consumes  more  time 


URINE  199 

than  that  of  Hopkins ,  but  it  yields  more  accurate  and 
useful  results. 

6.  Estimation  of  Chlorides 

According  to  Mohr. 

Principle. — If  a  solution  of  sodium  chloride  is  treated 
with  a  little  potassium  chromate,  and  then  a  solution  of 
silver,  silver  chloride  is  precipitated.  After  all  the  chlo- 
rine has  combined  with  silver,  further  addition  of  the  sil- 
ver solution  produces  silver  chromate,  which  colors  the 
precipitate  orange. 

NECESSARY  SOLUTIONS. — 1.  Silver  Solution. — This  is 
made  by  dissolving  29.042  grammes  of  pure  silver  nitrate 
in  a  litre  of  distilled  water. 

2.   A  10  per  cent,  solution  of  potassium  chromate. 

Procedure. — Ten  cc  of  urine  are  diluted  in  a  flask  or 
beaker  with  30  to  50  cc  of  distilled  water,  and  treated  with 
a  few  drops  of  the  potassium  chromate  solution,  until  a 
distinct  yellow  coloration  is  produced. 

The  silver  solution  is  then  run  in  from  a  burette,  with 
vigorous  agitation,  until  the  reddish  coloration  no  longer 
disappears  as  at  first.  One  cc  of  the  silver  solution  repre- 
sents 0.01  gramme  of  sodium  chloride. 

This  method  gives  results  sufficiently  accurate  for 
clinical  and  practical  use;  more  accurate  results  are  ob- 
tained if  the  urine  is  .reduced  to  ashes,  and  the  chlorine 
in  the  ashes  estimated  according  to  the  same  method. 

7.  Estimation  of  Phosphates 

Volumetric  Analysis. 

Principle. — If  phosphates  in  a  hot  acetic  acid  solution 
are  brought  in  contact  with  a  solution  of  uranium  nitrate, 
the  phosphoric  acid  is  entirely  precipitated  as  uranium 
phosphate. 


200    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

NECESSARY  SOLUTIONS. —1.  A  Solution  of  Sodium 
Acetate  in  Acetic  Acid. — One  hundred  grammes  of  sodium 
acetate  are  dissolved  in  800  cc  of  water,  100  cc  of  30  per 
cent,  acetic  acid  added,  and  the  solution  brought  up  to  a 
litre. 

2.  Uranium  Nitrate  Solution. — This  solution  contains 
about  14  grammes  of  uranium  nitrate  to  a  litre  of  water, 
and  is  made  with  an  accurately  prepared  solution  of 
sodium  phosphate,  which  contains  0. 1  P2  05  in  50  cc. 
One  cc  of  this  solution  represents  0.005  gramme  of  phos- 
phoric acid. 

8.  A  10  per  cent,  solution  of  potassium  ferrocyanide. 

Procedure. — Fifty  cc  of  urine  in  an  Erlenmeyer  flask 
are  treated  with  5  cc  of  the  acetic  acid  sodium  acetate 
solution,  and  heated  to  the  boiling-point.  The  uranium 
nitrate  solution  is  now  run  in  as  long  as  the  formation  of 
a  precipitate  can  be  distinctly  seen;  a  drop  of  the  liquid 
is  then  tested  with  potassium  ferrocyanide  after  the  addi- 
tion of  each  J  cc  to  determine  if  the  end-reaction  has 
taken  place.  For  this  purpose  a  series  of  drops  of  the 
potassium  ferrocyanide  solution  are  placed  on  a  porcelain 
dish,  and  a  drop  removed  from  the  solution  with  a  glass 
rod  is  allowed  to  run  into  one  of  them.  If  a  reddish- 
brown  coloration  appears  at  the  point  of  contact  of  the  two 
drops,  the  end-reaction  has  taken  place  (potassium  ferro- 
cyanide combines  with  the  uranium,  forming  uranium 
ferrocyanide,  which  forms  a  reddish-brown  precipitate). 
The  number  of  cc  of  the  uranium  solution  used,  multiplied 
by  0.005,  gives  the  amount  of  P205  in  50  cc  of  urine. 
Instead  of  potassium  ferrocyanide,  tincture  of  cochineal 
may  be  used  as  indicator;  the  hot  solution  is  treated  with 
1  to  2  cc  of  the  tincture,  and  titrated  with  uranium  nitrate 
until  it  becomes  grass-green.  The  urine  must  be  free  from 
albumin.  The  method  gives  good  results. 


URINE  201 

8.  Estimation  of  Sulphates. 

Sulphuric  acid  appears  in  the  urine  in  two  forms,  in 
the  form  of  sulphuric  acid  salts  (  =  preformed  sulphates) , 
and  in  combination  with  aromatic  alcohols,  as  phenol, 
indoxyl,  and  brenzkatechin  (—  O—  dihydroxylbenzol  = 
C6H4[OH]2)  as  sulphuric  acid  esters  or  combined  sul- 
phuric acid,  j 

(a)  Estimation  of  the  Preformed  Sulphates. 

Principle. — The  sulphuric  acid  is  precipitated  in  an 
acid  solution  with  barium  chloride,  and  estimated  gravi- 
metrically. 

Procedure. — Fifty  to  one  hundred  cc  of  the  filtered 
urine  are  diluted  with  an  equal  quantity  of  [water,  acidi- 
fied with  acetic  acid,  treated  with  an  excess  of  barium 
chloride  solution,  and  heated  on  a  water-bath  until  the 
precipitate  of  barium  sulphate  has  settled.  The  precipitate 
is  then  collected  on  an  ash-free  filter,  washed  with  hot 
water  until  it  is  entirely  free  from  chlorine  (no  clouding 
upon  the  addition  of  silver  nitrate  and  nitric  acid) ,  the 
filter  and  precipitate  then  dried,  and  reduced  to  ashes  in 
a  platinum  crucible.  After  it  has  cooled,  the  platinum 
crucible  and  the  ashes  are  weighed,  the  weight  of  the 
crucible  subtracted,  and  the  difference  multiplied  by 
0.34331.  The  product  represents  the  S03  contained  in 
the  urine  used. 

(/')  Estimation  of  the  Combined  Sulphuric  Acid. 

The  filtrate  from  the  above -estimation  is  strongly 
acidified  with  hydrochloric  acid  (by  the  addition  of  10 
cc  of  IIC1) ,  boiled  for  some  time,  and,  if  necessary,  a  few 
drops  of  a  hot  solution  of  barium  chloride  are  added. 
The  combined  sulphuric  acid  is  liberated  by  the  boiling 
with  hydrochloric  acid,  and  precipitated  as  a  barium  salt. 
The  precipitate  is  collected  upon  an  ash-free  filter,  and 


202    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

the  further  estimation  carried  out  as  in  estimating  the 
preformed  sulphates. 

If  the  results  of  both  estimations  are  added,  the  total 
sulphuric  acid  is  obtained.  This  is,  in  the  normal  adult, 
1.5  to  3  grammes  S03  in  the  twenty- four  hours7  sample  of 
urine. 

9.  Estimation  of  Oxalic  Acid  According  to  Salkowski 

Principle. — The  oxalic  acid  is  extracted  with  alcohol 
and  ether  from  urine  containing  hydrochloric  acid :  after 
distillation  of  the  alcohol  and  ether,  the  oxalic  acid  is 
precipitated  as  a  calcium  salt,  and  estimated  gravimetri- 
cally. 

Procedure. — Five  hundred  cc  of  unfiltered  urine  are 
evaporated  down,  on  a  water-bath,  to  about  150  cc,  treated, 
after  cooling,  with  20  cc  of  concentrated  hydrochloric  acid, 
and  placed  in  a  500  cc  separatory  funnel.  The  mixture 
is  shaken  three  times  with  an  equal  quantity  of  alcohol 
and  ether  (nine  parts  ether,  one  part  absolute  alcohol), 
and  the  ether  extract  is  collected  in  a  flask.  The  ether 
extract  is  then  filtered  through  a  dry  filter  into  a  dry  dis- 
tilling-flask,  and  the  ether  distilled.  The  fluid  remain- 
ing in  the  flask  is  poured  into  a  porcelain  dish,  and  the 
flask  is  rinsed  first  with  alcohol,  and  then  with  water, 
pouring  these  also  into  the  dish.  The  dish  is  heated  on 
a  water-bath  (a  little  water  being  added)  until  the  odor  of 
alcohol  and  ether  has  disappeared.  The  watery  solution 
which  remains  (its  volume  should  be  about  20  cc)  throws 
down  the  resinous  substances  en  cooling.  The  solution 
is  filtered,  the  filtrate  rendered  slightly  alkaline  with  am- 
monia, treated  with  1  to  2  cc  of  a  10  per  cent,  solution  of 
calcium  chloride,  and  acidified  with  acetic  acid.  The 
solution  is  then  set  aside  in  a  warm  place  for  some  time 
(twelve  to  twenty-four  hours)  until  the  precipitate  of  cal- 


URINE  203 

cium  oxalate  has  collected  on  the  bottom  of  the  receptacle. 
The  precipitate  is  then  collected,  without  loss,  on  an  ash- 
free  filter,  washed  with  water,  dried,  thoroughly  burned 
(to  convert  the  calcium  oxalate  into  caustic  lime),  and 
weighed.  The  weight  of  the  caustic  lime  (CaO)  multi- 
plied by  f  gives  the  amount  of  oxalic  acid.  If  the  esti- 
mation is  properly  carried  out,  the  caustic  lime  gives  off 
no  carbon  dioxide  when  dissolved  in  dilute  hydrochloric 
acid ;  the  solution  must  also  give  a  negative  reaction  when 
tested  for  phosphoric  acid  with  ammonium  molybdate. 
This  method  yields  accurate  results.  Normal  urine,  con- 
tains not  more  than  0.02  gramme  of  oxalic  acid  in  twenty- 
four  hours. 

10.  Schloesing's  Method  of  Determining  Ammonia 

Principle. — Ammonia  is  liberated  by  milk  of  lime  and 
taken  up  by  sulphuric  acid  in  a  closed  vessel. 

Solutions  Required. — 1.  One-quarter  normal  sulphuric 
acid.  2.  One  decinormal  solution  of  sodium  hydrate. 

Determination. — Twenty-five  cc  of  filtered  urine  are  put 
into  a  flat  dish,  the  walls  of  which  run  up  perpendicularly, 
and  this  dish  is  put  on  the  plate  of  a  large  exsiccator 
(this  consists  of  a  glass  plate  and  a  glass  bell  cover).  A 
triangle  made  of  glass  is  put  into  the  dish  and  on  it  is 
put  a  small  dish  into  which  are  added  20  cc  of  a  one- 
quarter  normal  sulphuric  acid  by  means  of  a  pipette.  To 
the  urine  are  added  at  least  10  cc  of  milk  of  lime  and  the 
glass  plate  is  now  covered  with  the  glass  bell,  the  border 
of  which  has  previously  received  a  coat  of  lard.  After 
three  to  four  days  almost  all  of  the  ammonia  has  been  ex- 
pelled and  absorbed  by  the  sulphuric  acid.  If  the  moisture 
which  settled  on  the  inner  wall  of  the  bell  reacts  alkaline, 
it  is  washed  into  the  sulphuric  acid.  .By  the  titration  with 
the  decinormal  sodium  hydrate  solution  is  determined  how 


204     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

much  of  the  ammonia  has  combined  with  the  sulphuric 
acid;  1.7  mg  ammonia  corresponds  to  1  cc  of  the  deci- 
normal  sulphuric  acid  solution  (20  cc  of  J  normal  acid 
corresponds  to  50  cc  ^  normal  acid)  .  In  this  titration 
methyl  orange  is  used  as  an  indicator. 

11.  Messinger's  Method  of  Determining  Acetone 

Principle.  —  In  an  alkaline  solution  acetone  is  changed 
into  iodoform  by  iodine-kalium  iodide.  The  iodine  does 
not  act  as  such  on  the  acetone,  but  as  an  iodide  which 
forms  by  the  reaction  of  the  iodine  upon  the  alkaline 
hydrate.  The  reaction  takes  place  as  follows  : 


CH3COCH3  -f  3KOI  =  CH3COCI3  +  3KOH 

Acetone         Kalium  iodid 

CH3COCI3+  KOH  =  CH3COOK  +  CHI3 

Iodoform 

In  the  transformation  of  one  molecule  of  acetone  into 
one  molecule  of  iodoform,  6  atoms  of  iodine  are  required. 
First  is  added  iodine  in  excess,  and  the  remaining  quan- 
tity of  iodine  is  then  determined  by  titration  with 
natrium-thio-sulphate  which  is  changed  into  tetra-thio- 
sodium. 

ONa  S02ONa 

2SO2  +I2=S2  +  2NaI 

SNa  S02ONa 

The  quantity  of  the  iodine  is  calculated  from  the  quan- 
tity of  the  iodine  in  combination. 

Solutions  Required.  —  1.  One-tenth  normal  solution  of 
iodine.  2.  One-tenth  normal  solution  of  thio-sulphate. 
8.  Fifty  per  cent,  solution  of  acetic  acid.  4.  Sulphuric 
acid  diluted  eight  times.  5.  A  starch  solution. 


URINE  205 

Determination. — The  acetone  is  distilled  off  the  urine. 
The  distillate  must  not  contain  either  phenol,  or  ammoniaj 
or  nitrous  acid,  or  formic  acid.  One  hundred  cc  of  urine 
are  distilled  together  with  2  cc  of  a  50  per  cent,  solution 
of  acetic  acid  (acetic  acid  keeps  back  the  phenol).  This 
distillate  is  again  distilled  with  1  cc  cf  the  sulphuric  acid 
solution  (solution  No.  4,  above),  ammonia  combining 
with  the  sulphuric  acid.  Each  distillation  is  continued 
until  only  one-fourth  of  the  solution  remains. 

Into  a  flask,  having  a  good  fitting  glass  stopper,  is 
put  the  second  distillate  to  which  is  added  in  excess  the 
iodine  solution  (2)  (50  to  100  cc)  ;  the  flask  is  shaken  up, 
and  then  is  added  drop  by  drop  in  excess  a  concentrated 
solution  of  sodium  hydrate  which  is  free  from  nitrites. 
The  iodine  color  disappears  and  iodoform  settles  at  the 
bottom.  The  flask  is  now  stoppered,  well  shaken  up  and 
left  for  five  minutes.  The  fluid  is  then  acidulated  with 
concentrated  hydrochloric  acid,  whereby  the"  liberated 
iodine  separates.  The  titration  with  the  thio-sulphate 
solution  is  continued  until  the  dark  brown  color  has 
changed  to  a  faint  yellow;  now  a  few  €C  of  the  starch 
solution  are  added.  There  appears  at  first  a  green  or  a 
brownish  green  color;  on  the  addition  of  more  thio- 
sulphate  a  pure  blue  color  results.  The  titration  is  con- 
tinued until  the  fluid  is  entirely  decolorized;  toward  the 
end  the  thio-sulphate  i,s  added  drop  by  drop.  Each  cc 
of  the  iodine  solution  in  combination  corresponds  to 
0.967  mg  of  acetone. 

Illustration. — Assuming  100  cc  of  the  iodine  solution 
have  been  added  and  in  the  titration  28  cc  of  thio-sulphate 
have  been  used.  Since  1  cc  of  the  thio-sulphate  solution 
corresponds  to  1  cc  of  the  iodine  solution,  therefore  100 
—  28  cc  =  77  cc  of  the  iodine  solution  were  used  to  form 
the  iodoform. 


206     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Therefore  1  litre  contains  77x0.967  mg=74.459  mg 
in  100  cc  of  urine  or  0.74459  gr  to  a  litre  of  acetone. 

VI.  Examination  of  Urinary  Calculi  and  Concretions 

According  to  their  composition,  the  following  varieties 
are  distinguished: 

1.  Uric  acid  stones,  which  are  -composed  of  free  uric 
acid,  acid  sodium  urate,   or   (more  rarely)   ammonium 
urate. 

2.  Phosphatic  stones,  which  are  composed  principally 
of  phosphates  of  calcium,  magnesium,  and  calcium'  car- 
bonate. 

8.  Oxalate  stones,  composed  of  calcium  oxalate. 

4.  Cystin  and  xanthin  stones  (very  rare  concretions) . 

5.  Mixed  stones,  which  are  composed  of  layers  of  vary- 
ing composition. 

General  Characteristics 

Color, — Uric  acid  stones  are  yellow  to  dark  brownish- 
red;  phosphatic  stones  are  whitish,  grayish  to  grayish- 
yellow;  oxalate  stones  usually  brownish-red  to  black, 
though  occasionally  they  are  white  or  gray  (the  smaller 
stones)  ;  cystin  stones  are  pale  yellow ;  xanthin  stones  light 
brown. 

Surface. — Oxalate  stones  have  a  rough,  irregular,  or 
warty  surface  (mulberry  stones);  uric  acid  stones  a  less 
rough;  phosphatic  stones  usually  a  sandy,  comparatively 
smooth  surface;  cystin  and  xanthin  stones  are  usually 
smooth. 

Consistency. — The  softest  stones  are  the  cystin  and  phos- 
phatic stones ;  the  latter  are  more  or  less  earthy  or  chalky, 
and  comparatively  crumbly  in  consistency.  Cystin  stones 
are  of  waxy  softness,  uric  acid  stones  much  harder,  and 
oxalate  stones  are  the  hardest. 


URINE  207 

Chemical  Examination 

For  examination  the  stones  are  sawn  into  two  equal 
parts,  the  cut  surface  smoothed  a  little,  and  washed  with 
water.  The  strata,  of  which  the  stone  is  composed,  and 
the  nucleus  then  appear  distinct.  For  chemical  exami- 
nation, a  portion  is  scraped  from  each  stratum  and  from 
the  nucleus,  and  each  portion  is  examined  separately. 
If  no  stratification  and  no  nucleus  are  seen  on  the  cut  sur- 
face, the  stone  is  crushed  and  rubbed  to  a  fine  powder  in 
a  mortar.  A  small  portion  of  the  powder  is  burned  on  a 
platinum  spatula.  This  preliminary  test  determines  the 
course  of  further  chemical  examination,  since  the  excess 
of  organic  or  inorganic  matter  in  the  stone  is  determined 
by  it.  Two  things  may  happen : 

1.  Almost  all  the  material  burns  up  and  very  little  or 
no  residue  is  left — i.e.,  the  stone  is  composed  principally 
of  organic  matter.  Such  stones  may  be  composed  of  uric 
acid,  urates,  xanthin,  or  cystin.  Uric  acid  and  xanthin 
stones  burn  without  flame,  and  with  an  odor  suggesting 
hydrocyanic  acid;  cystin  stones  with  a  bluish  flame,  and 
an  odor  suggesting  sulphurous  acid. 

To  determine  which  of  the  above-mentioned  organic 
substances  constitutes  the  main  portion  of  the  stone,  a 
second  portion  is  evaporated  to  dryness  in  a  porcelain  dish 
with  a  few  drops  of  nitric  acid.  If  the  residue  turns 
purple-red  on  the  addition  of  a  drop  of  ammonia,  and 
blue-violet  on  the  addition  of  a  drop  of  sodium  hydrate 
(murexide  test),  it  is  composed  of  uric  acid,  ammonium 
urate,  or  some  other  urate.  If  the  original  substance 
liberates  ammonia  upon  the  addition  of  potassium  hydrate, 
the  stone  is  composed  of  ammonium  urate.  If  the  test 
for  ammonia  is  negative,  and  the  stone  burns  up  com- 
pletely, it  is  composed  of  pure  uric  acid.  Other  urates 
leave  a  slight  residue  on  burning. 


208    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

If  with  the  murexide  test  no  coloration  is  produced 
with  ammonia,  while  with  sodium  hydrate  a  beautiful  red 
coloration  is  produced,  the  stone  is  composed  of  xanthin. 
Cystin  stones  give  a  negative  result  with  the  murexide 
test,  both  with  ammonia  and  sodium  hydrate.  They  are 
distinguished  by  the  fact  that  they  are  readily  soluble  in 
ammonia,  and  that  when  the  ammonia  is  slowly  evapor- 
ated, the  cystin  crystallizes  in  very  characteristic  hexagonal 
plates. 

2.  The  material  does  not  burn  at  all,  or  merely  turns 
black,  and  leaves  a  very  marked  residue  when  burned. 
In  this  case,  the  stone  may  be  principally  composed  of 
phosphates,  carbonates,  or  oxalates.  A  small  portion  is 
slightly  heated  with  dilute  hydrochloric  acid,  whereupon 
the  majority  of  the  powder  is  dissolved.  Only  the  organic 
basic  substance,  and  any  uric  acid  which  may  be  present, 
remain  undissolved.  The  solution  is  allowed  to  cool  (in 
order  to  precipitate  the  uric  acid),  filtered,  the  filtrate 
diluted  with  water,  and  rendered  strongly  alkaline  with 
ammonia.  If  a  precipitate  is  produced  by  the  addition 
of  ammonia,  it  may  consist  of : 

(a)  Earthy  phosphates  (calcium  and  magnesium  phos- 
phates) ,  triple  phosphates  (ammonium  magnesium  phos- 
phate) ;  or, 

(b)  Calcium  oxalate. 

The  precipitate  is  separated  from  the  solution  (best  by 
centrifugalization)   and  dissolved  in   acetic   acid.      The] 
triple  phosphates  and  earthy  phosphates  are  thus  dissolved, 
while  calcium  oxalate  remains  undissolved,  and  can  be 
detected  microscopically. 

The  following  test  is  carried  out  with  the  filtered  acetic 
acid  solution.  It  is  treated  with  ammonium  molybdate 
and  nitric  acid,  and  heated  to  60°  C.  If  a  yellow  precipi-i 
tate  is  formed,  phosphoric  acid  is  present. 


URINE  209 

If  upon  the  addition  of  ammonia  to  the  hydrochloric 
acid  solution  of  the  stone  no  precipitate  is  formed,  it  may 
be  composed  of  calcium  or  magnesium  carbonate. 

A  portion  of  the  stone  is  then  touched  with  hydro- 
chloric acid,  by  which  gas  (C02)  will  be  liberated.  One- 
half  of  the  ammoniacal  solution  is  treated  with  ammonium 
oxalate;  if  a  precipitate  of  calcium  oxalate  is  produced, 
calcium  carbonate  is  present.  To  the  other  half  a  solution 
of  sodium  phosphate  is  added;  if  a  precipitate  of  triple 
phosphate  is  produced,  magnesium  carbonate  is  present. 

The  portion  of  the  stone  undissolved  by  .hydrochloric 
acid  must  be  tested  for  uric  acid  with  the  murexide  test. 

VII.  Microscopical  Examination  of  the  Urinary 
Sediment 

There  are  three  methods  for  collecting  urinary  sediment 
for  microscopical  examination : 

1.  Sedimentation   in  a    Conical   Glass. — The    urine    is 
allowed  to  stand  undisturbed  for  some  time  in  a  conical 
glass;  solid  insoluble  constituents  gradually  sink  and  col- 
lect as  a  precipitate  at  the  apex  of  the  glass.     After  the 
solution  has  been  decanted  as  completely  as  possible,  a  drop 
of  the  sediment  is  removed  with  a  pipette  for  examination. 

2.  Collection  of  the  Precipitate  on  a  Filter. — As  large  a 
quantity  as  possible  of  the  urine  to  be  examined  is  filtered 
through  a  moist  filter,  upon  which  the  solid  constituents 
collect. 

3.  Precipitation  of  the  Solid  Constituents  by  Centrifugali- 
zation  of  the  Urine. — A  small  glass  tube  with  a  conical 
bottom  is  filled  with  the  urine,1  placed  in  the  holder  of  a 

1  It  is  advisable  to  allow  the  urine  to  stand  one  to  two  hours 
before  taking  the  portion  to  be  examined,  and  then  to  remove  the 
lowest  portion  with  a  long  pipette,  and  centrifugalize  it. 


210    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

centrifuge,  and  centrifugalized  for  a  few  minutes.  The 
insoluble  constituents  are  thus  collected  at  the  apex  of  the 
centrifuge-tube.  The  liquid  above  the  sediment  is  poured 
off  by  inverting  the  tube  as  quickly  as  possible.  The 
liquid  should  not  be  poured  off  gradually,  as  the  sediment 
is  then  apt  to  become  mixed  with  it. 

The  advantages  of  centrifugalization  are  evident.  By 
sedimentation  in  a  conical  glass  urine  containing  but  few 
solid  constituents  yields  almost  no  precipitate,  while  when 
centrifugalized  it  yields  sufficient  sediment  for  examina- 
tion. Moreover,  with  the  latter  method  it  is  not  necessary 
for  the  urine  to  stand  for  hours,  during  which  decomposi- 
tion, and  therefore  alteration  of  its  solid  constituents, 
easily  take  place.  A  drop  of  the  sediment  is  removed 
with  a  pipette,  placed  on  a  slide,  and,  without  pressure, 
is  covered  with  a  cover-glass.  If  the  solution  extends  be- 
yond the  cover-glass,  the  excess  must  not  be  absorbed  with 
filter-paper,  since  by  so  doing  the  solid  constituents  might 
be  drawn  out  from  under  the  cover-glass  with  the  fluid. 
It  is  then  examined  microscopically  with  a  magnification 
of  300  to  400.  As  is  always  the  case  in  examining  un- 
stained objects,  the  concave  mirror  is  used,  and  the  Able 
condenser  thrown  out. 

Microchemical  reactions  must  frequently  be  used  for 
the  identification  of  amorphous  and  crystalline  salts. 
These  are  carried  out  by  placing  a  drop  of  the  reagent  at 
one  side  of  the  cover-glass,  and  drawing  it  through  under 
the  glass  by  means  of  a  piece  of  filter-paper,  which  is 
placed  at  the  opposite  side. 

Microscopical  Examination 

Urinary  sediment  is  composed  of  unorganized  and 
organized  solids. 

The  unorganized  constituents  are  the  salts  which  are 


URINE  -  211 

precipitated  from  the  urine,  and  which  appear  in  the  sedi- 
ment either  in  amorphous  or  crystalline  form. 

No  attempt  should  be  made  to  divide  the  salts  accord- 
ing to  the  reaction  of  the  urine  in  the  sediment  of  which 
they  are  usually  found,  since  most  of  them  may  be  contained 
in  the  sediment  of  acid,  amphoteric,  and  alkaline  urine. 
Uric  acid,  for  example,  appears  principally  in  acid,  am- 
monium magnesium  phosphate  in  alkaline  urine;  never- 
theless, they  may  appear  together  in  alkaline  urine,  when, 
in  the  early  stage  of  alkaline  fermentation,  the  uric  acid 
crystals  are  not  yet  completely  dissolved,  while  the  triple 
phosphate  crystals  have  already  been  precipitated.  In  the 
description  of  the  various  salts  the  reaction  of  the  urine 
in  which  they  are  usually  found  will  be  mentioned. 

Uric  Acid  (Plate  V,  Fig.  I).— Crystals  of  uric  acid 
appear  principally  in  the  sediment  of  acid  urine,  more 
rarely  in  that  of  amphoteric,  and  only  under  special  con- 
ditions in  that  of  alkaline  urine.  They  sometimes  appear 
singly,  and  sometimes  in  great  quantity,  and  then  fre- 
quently cling  to  the  sides  and  bottom  of  the  vessel,  and 
can  usually  be  recognized  macroscopically  by  their  crystal- 
line appearance  and  their  yellow  or  red-brown  color. 

Microscopically,  uric  acid  crystals  appear  almost 
always  brown  or  yellow;  colorless  crystals  are  very  rarely 
seen.  They  vary  greatly  in  form  and  size.  They  appear 
in  the  form  of  whetstones  and  of  spindles,  which,  lying 
crosswise  over,  each  other,  resemble  glands  and  rosettes, 
as  hexagonal  plates,  and  in  cask  or  barrel  forms.  Spear 
and  needle  forms  are  also  seen,  arranged  in  sheaves  or 
tufts.  Dumb-bell  and  hour-glass  forms  are  more  rarely 
seen.  These  various  forms  of  crystals,  which  frequently 
appear  side  by  side,  can  always  be  traced  back  to  a  com- 
mon form — the  rhomboid  plate.  If  two  opposite  angles 
of  the  plate  are  rounded,  the  whetstone  form  is  produced; 


212    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

if  they  are  cut  off  by  straight  lines,  the  hexagonal  forms 
are  produced;  if  the  corners  are  drawn  out  to  an  angle, 
the  needle  or  spear  forms  are  produced;  if  the  crystals  are 
piled  upon  one  another,  the  cask  and  barrel  forms  are  pro- 
duced. 

Uric  acid  crystals  can  usually  be  recognized  at  once  by 
their  color,  which  they  owe  to  the  urinary  pigments 
extracted  at  the  time  of  their  crystallization.  The  color- 
less four  to  six  sided  plates,  in  which  uric  acid  may  crys- 
tallize, resemble  cystin  crystals,  but  can  be  distinguished 
from  them  by  their  chemical  behavior. 

Microcliemical  Reactions : 

1.  Uric  acid  gives  the  murexide  test  (cf.  page  207). 

2.  If  a  little  sodium  hydrate  is  allowed  to  run  under 
the  cover-glass,  the  crystals  of  uric  acid  are  dissolved,  to 
be  reprecipitated  upon  the  addition  of  hydrochloric  acid. 

Amorphous  Urates  (Fig.  25). — These  consist  of  the 
urates  of  sodium,  potassium,  calcium,  and  magnesium, 
and  form  a  sediment  of  acid  urine.  Microscopically, 
they  appear  as  a  clay-colored,  yellow,  or  brick-red  sedi- 
ment, which  is  often  precipitated  in  large  quantity  from 
concentrated  acid  urine,  and  from  urine  exposed  to  the 
cold  (sedimentum  later  itiutii) .  The  color  of  this  sediment 
is  due  to  the  normal  pigments  of  the  urine,  which  the 
urates,  like  uric  acid,  extract  when  precipitated. 

Microscopically,  they  appear  as  small,  amorphous, 
brownish-yellow,  more  rarely  colorless  granules,  which 
usually  lie  together  in  mosslike  groups  of  varying  size, 
often  in  such  thick  masses  that  they  cover  the  entire  field, 
hiding  all  other  solid  elements.  To  render  these  latter 
visible  it  is  necessary  to  dissolve  the  urates.  This  is  most 
easily  accomplished  by  filling  the  centrifuge-tube,  con- 
taining the  sediment,  with  lukewarm  physiological  salt 
solution,  dissolving  the  urates  by  shaking,  and  centrifu- 


URINE  213 

galizing  at  once,  before  they  can  be  reprecipitated  by  the 
cooling  of  the  mixture.  Occasionally  urates  form  peculiar 
cylindrical  figures,  urate  casts,  which  must  not  be  confused 
with  granular  casts ;  frequently  they  are  seen  lying  upon 
epithelial  cells  and  true  casts. 


FIG.  25.— a,  Urate  casts;  6,  neutral  calcium  phosphate. 

Microchemical  Reactions : 

1.  Urates  are  dissolved  by  heating,  and  reprecipitated 
by  cooling. 

2.  They  are  dissolved  by  the  addition  of  hydrochloric 
and  acetic  acids,   uric  acid  crystals  being  after  a  time 
precipitated  from  the  solution  in  the  form  of  rhomboid 
plates. 

8.  The  murexide  test  is  positive. 

Ammonium  Urate    (Plate    VI,    Fig.  J). — Ammonium 
urate  is  the  only  salt  of  uric  acid  which  is  found  in  the 


214    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

sediment  of  alkaline  urine.  It  is  found  rather  frequently 
in  neutral  and  acid  urine  in  children,  especially  newborn 
and  nursing  children,  much  more  rarely  in  adults. 

The  presence  of  ammonium  urate  cannot  be  detected 
macroscopically.  Its  microscopical  appearance  is,  how- 
ever, very  characteristic ;  usually  it  appears  in  the  form  of 
brownish-yellow  spheres,  which  may  lie  singly,  in  pairs, 
or  in  large  groups.  These  spheres  frequently  show 
spicules,  which,  according  to  their  size  and  number,  give 
a  varied  appearance  to  the  crystals.  Thus,  crystals  of 
thorn-,  apple-,  mace-,  mite-,  and  turnip-form  are  pro- 
duced. Crystals  of  ammonium  urate  are  rarely  colorless.. 
They  then  appear  as  dumb-bells  or  as  tufts  of  needles. 
The  simultaneous  appearance  of  typical  brown  spheres,  as 
well  as  microchemical  reactions,  make  it  possible  to  easily 
recognize  these  rarer  crystals. 

Microchemical  Reactions : 

1.  Crystals  of  ammonium  urate  are  dissolved  by  heat- 
ing, and  are  reprecipitated  by  cooling. 

2.  Upon  the  addition  of  acetic  acid  they  are  dissolved, 
and  in  their  place  crystals  of  uric  acid  are  formed. 

8.  They  are  dissolved  by  potassium  hydrate  with  the 
formation  of  gas  (ammonia) . 

4.   Like  all  urates,  they  give  the  murexide  reaction. 

Calcium  Oxalate  (Fig.  26). — Crystals  of  calcium  oxal- 
ate  appear  in  the  sediment  of  acid,  amphoteric,  and  faintly 
alkaline  urine.  When  precipitated  in  large  quantity, 
they  form  a  grayish-white,  flaky  sediment.  They  appear 
usually  as  colorless,  highly  refractive  octahedra,  the  so- 
called  envelope- forms,  of  varying  size.  Very  small  crys- 
tals, whose  envelope-form  can  often  be  detected  only  by 
careful  focusing,  are  seen,  especially  when  calcium  oxalate 
is  precipitated  in  large  quantity.  Even  the  most  minute, 
punctiform  crystals  attract  attention,  however,  by  their 


URINE  215 

characteristic  glistening  appearance,  often  resembling 
minute  fat  drops,  from  which  they  are  distinguished  by 
microchemical  reactions.  (Fat  is  dissolved  by  the  addi- 
tion of  ether.) 

Calcium  oxalate  crystallizes  in  hour-glass,  dumb-bell, 
or  biscuit  form,  more  rarely  than  in  octahedra.  The  high 
refractive  power  of  these  objects,  whose  surface  is  slightly 


FIG.  26.— Calcium  Oxalate. 

striated,  the  simultaneous  presence  of  envelope  forms,  and, 
finally,  their  behavior  toward  chemical  reagents,  make  it 
possible  to  recognize  them  as  crystals  of  calcium  oxalate. 
In  icteric  urine  they  are,  like  other  solid  constituents 
(epithelium  cells,  casts,  etc. ) ,  frequently  yellow. 

Crystals  of  calcium  oxalate  are  characterized  chemically 
by  their  insolubility  on  heating,  and  in  acetic  acid,  and 
their  easy  solubility  in  hydrochloric  acid.  If  ammonia 


216     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

or  potassium  hydrate  is  added  to  the  solution  in  hydro- 
chloric acid,  the  calcium  oxalate  recry stall izes  in  octa- 
hedra. 

Neutral  Calcium  Phosphate  (Dicalcium  Phosphate — Fig. 
27). — This  appears  in  the  sediment  of  slightly  acid,  am- 
photeric,  and  faintly  alkaline  urine.  Neutral  calcium 
phosphate  crystallizes,  usually  in  long,  glistening,  pris- 


FlG.  27.— a.  Crystals  of  neutral  calcium  phosphate; 
b,  amorphous  phosphates  and  carbonates. 

matic,  cuneiform  crystals,  which  may  be  seen  singly,  but 
are  usually  arranged  in  bundles  of  varying  thickness,  or 
in  rosettes.  In  the  latter  case  the  arrow-pointed  heads  are 
usually  directed  toward  the  centre.  Dicalcium  phosphate 
also  crystallizes  in  plates,  and,  in  rare  cases,  in  tufts  of 
needles,  which  resemble  tyrosin  crystals  in  appearance, 
but  can  be  distinguished  from  them  by  their  microchemi- 


URINE  217 

cal  reactions.  Crystals  of  neutral  calcium  phosphate  are 
completely  dissolved  by  treating  with  acetic  acid. 

Calcium  Sulphate. — Crystals  of  calcium  sulphate  are  very 
rarely  detected  in  the  sediment  of  the  urine.  They  appear 
only  in  highly  acid  urine,  in  which,  if  present,  they  fre- 
quently form  a  thick  white  precipitate. 

Microscopically,  they  appear  as  long,  colorless  needles, 
or  as  slim  prisms,  with  oblique  bases,  usually  arranged 
in  rosettes.  The  following  microchemical  reaction  pre- 
vents confusion  with  the  crystals  of  neutral  calcium  phos- 
phate, which  resemble  them  closely.  Crystals  of  calcium 
sulphate  are  insoluble  in  acetic  acid,  and  soluble  with 
difficulty  in  hydrochloric  acid. 

Calcium  Carbonate. — Calcium  carbonate  appears  most 
frequently  in  the  sediment  of  alkaline,  much  less  fre- 
quently in  that  of  amphoteric  or  faintly  acid  urine.  It 
usually  appears  'together  with  amorphous  phosphates, 
from  which  it  cannot  be  distinguished  macroscopically. 
Microscopically,  it  appears  in  the  form  of  small  grayish- 
white  granules  or  spherules,  which  frequently  lie  upon 
one  another.  Their  microchemical  behavior  is  charac- 
teristic. Upon  the  addition  of  dilute  mineral  acids,  the 
carbonates  are  dissolved  with  the  liberation  of  CO2,  so 
that  the  entire  microscopical  field  is  covered  with  minute 
air-bubbles. 

Amorphous  Earthy  Phosphates  (Calcium  and  Magnesium 
Phosphates — cf.  Fig.  27). — These  are  most  frequently 
precipitated  from  alkaline,  but  may  appear  in  the  sedi- 
ment of  amphoteric  or  faintly  acid  urine.  They  form  a 
fine,  flaky,  grayish-white,  easily  mobile  precipitate. 

Microscopically,  they  appear  as  finely  granular,  color- 
less masses,  which  can  be  easily  distinguished  by  micro- 
chemical  reactions  from  other  amorphous  sediments  resem- 
bling them  in  appearance.  The  earthy  phosphates  are 


218     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

dissolved  upon  the  addition  of  acetic  acid  without  the 
liberation  of  gas,  but  are  not  dissolved  by  heating. 

Ammonium  Magnesium  Phosphate  (Triple  Phosphate — 
Fig.  28). — This  appears  principally  in  the  sediment  of 
alkaline  urine,  frequently  together  with  amorphous  phos- 
phates and  carbonates,  as  well  as  in  the  purulent  sediment 
of  alkaline  urine.  It  is,  however,  occasionally  found  in 


FlG.  28. — Triple  Phosphate  (Ammonium  Magnesium 
Phosphate). 

amphoteric  and  faintly  acid  urine  in  commencing  alkaline 
fermentation. 

Triple  phosphate  forms  rhomboid,  clear  prisms  of  very 
characteristic  appearance.  Usually  they  appear  in  the 
coffin-lid  form,  more  rarely  as  penniform  or  fernlike 
structures.  Now  and  then  very  grotesque  crystals  are  pro- 
duced by  combinations  of  these  forms,  which,  however, 


URINE  219 

can  be  identified  -as  triple  phosphate  microchemically,  by 
their  easy  solubility  upon  the  addition  of  acetic  acid. 
This  characteristic  reaction  prevents  confusion  of  triple 
phosphate  crystals  with  the  large  envelope  forms  of  calcium 
oxalate,  which  occasionally  resemble  them  closely. 

Magnesium  Phosphate  Crystals. — These  are  found  in  very 
rare  cases  in  the  sediment  of  alkaline  urine  in  the  form  of 
glistening,  elongated,  rhomboid  plates,  which  are  easily 
soluble  in  acetic  acid.  They  are  also  frequently  seen  in 
the  film  which  covers  the  surface  of  alkaline  urine. 

Leucin  and  Tyrosin  (Fig.  29),  which  are  usually  found 
together,  do  not,  in  contradistinction  to  the  above- 


0 
FIG.  29.— a,  Tyrosin ;  b,  cystin ;  c,  leucin. 

described  forms  of  crystals,  appear  in  normal  urine. 
Their  appearance  has  been  observed  in  acute  yellow  atrophy 
of  the  liver,  phosphorus-poisoning,  and,  more  rarely,  in 
infectious  diseases,  as  typhoid  and  variola,  and  in  serious 
diseases  of  the  blood. 

The  detection  of  leucin  crystals  succeeds,  as  a  rule, 
only  after  the  evaporation  of  the  urine,  and  their  precipi- 
tation with  alcohol.  In  cases,  however,  in  which  leucin 
is  present  in  great  quantity,  it  crystallizes  spontaneously 
if  a  drop  of  the  urine  is  allowed  to  slowly  evaporate  on  a 
slide.  Tyrosin  is  soluble  with  more  difficulty  than  leucin, 
and  is  also  usually  present  in  the  urine  in  greater  quan- 
tity. It  is  often  precipitated,  therefore,  spontaneously 
after  the  urine  has  stood  awhile.  Tyrosin  crystals,  which, 


220     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

like  those  of  leucin,  are  usually  greenish-yellow,  form 
tufts  composed  of  very  fine  needles,  and  leucin  crystals 
form  spheres,  which  usually  allow  both  a  radial  and  con- 
centric striation  to  be  seen.  Small  spheres  are  frequently 
seen  attached  to  the  large  ones. 

Microcliemical  Reactions. — Leucin  is  soluble  in  acids 
and  alkalies,  insoluble  in  alcohol  and  ether.     Crystals  of 


FIG.  30.— Cystin  Crystals. 

ammonium  urate,  with  which  leucin  crystals  can  be  con- 
fused, are  distinguished  from  them  by  the  appearance  of 
uric  acid  crystals  after  they  have  been  dissolved  in  acetic 
acid. 

Tyrosin  is  insoluble  in  acetic  acid,  alcohol,  and  ether; 
soluble  in  dilute  mineral  acids,  alkalies,  and  ammonia. 

Cystin  (Fig.  30)  also  does  not  appear  in  the  urine 
under  normal  conditions.  It  appears  in  the  sediment  in 


URINE  221 

the  rare  cases  of  cystinuria,  in  which,  from  causes  not  yet 
thoroughly  explained,  cystin  is  excreted  in  the  urine. 
Cystin  crystallizes  in  characteristic,  colorless,  hexagonal 
plates,  which  are  frequently  arranged  in  strata.  Cystin 
is,  in  contradistinction  to  uric  acid,  soluble  in  hydro- 
chloric acid  and  ammonia;  it  is  insoluble  in  acetic  acid. 
If  acetic  acid  is  added  to  the  ammoniacal  solution,  or  if 
the  ammonia  is  allowed  to  slowly  evaporate,  the  cystin 
crystals  are  reprecipitated  in  the  form  of  hexagonal 
plates. 

Hippuric  Acid  (Fig.  81). — Crystals   of  hippuric   acid 
appear  very  rarely  in  the  sediment  of  the  urine.     Hippuric 


FIG.  31.— Crystals  of  Hippuric  Acid. 

acid  crystallizes  in  colorless  needles  and  rhomboid  prisms, 
which  may  have  a  stellate  arrangement.  It  is  insoluble 
in  acetic  acid. 

Cholesterin  appears  also  very  rarely  in  the  sediment  of 
the  urine.  Cholesterin  crystals  appear  as  colorless  plates, 
which  frequently  lie  in  strata,  and  have  notched  corners. 
For  microchemical  reactions,  cf.  p.  93. 

Xanthin,  though  normally  present  in  the  urine,  has  as 
yet  been  found  in  the  sediment  in  very  few  cases.  It  forms 
whetstone-shaped  crystals,  which  are,  in  contradistinction 
to  uric  acid,  readily  soluble  in  dilute  ammonia  and  on 
heating. 


222    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Of  the  -pigments  appearing  in  the  urine,  bile-pigment 
and  blood-pigment,  as  well  as  indigo,  occasionally  form 
amorphous  and  crystalline  precipitates. 

Bilirubin. — Bile-pigment  may  be  precipitated  as  orange- 
colored,  amorphous  granules,  or  as  yellow  crystals,  in  the 
form  of  needles  and  rhomboid  plates,  in  icterus  neonatorum 
and  icterus  gravis  of  adults,  especially  when  the  urine  is 
highly  acid.  The  granules  and  crystals  are  often  found 
in  epithelial  cells,  leucocytes,  or  fat  drops. 

Haemoglobin  (Plate  VII,  Fig.  K).— In  hemorrhage  from 
the  kidneys  and  the  urinary  passages  and  in  hsemoglobi- 
nuria,  blood-pigment  is  frequently  precipitated  in  the 
form  of  reddish  to  brownish-yellow  granules  and  flakes. 
Blood  pigment  may  be  precipitated  in  great  quantity, 
especially  in  severe  cases  of  ha3moglobinuria,  and  theni 
often  forms  cylindrical  objects  (pigment  casts) .  Blood- 
pigment  appears  more  rarely  in  the  form  of  hsematoidin 
crystals.  These  resemble  the  above-described  bilirubin 
crystals,  and  are  frequently  considered  as  identical  with 
them. 

Indigo- — Indigo-blue  is  occasionally  formed  in  alkaline 
decomposition  of  urine,  rich  in  indican,  by  the  oxidation 
of  the  indican.  The  blue  crystals,  often  noticeable 
macroscopically  because  of  their  color,  appear  as  small] 
rhomboid  plates  or  tufts  of  needles,  which  are  dissolved 
in  chloroform,  coloring  it  blue. 

Fat  and  Fatty- Acid  Needles  (Fig.  32).— When  fat  is 
found  in  the  urine  it  must  always  be  remembered  that  itsj 
presence  may  be  due  to  accidental  contamination,  by  means] 
of  greased  catheters,  suppositories,  greasy  receptacles,! 
etc.  Under  pathological  conditions  fat  appears  in  thej 
urine  in  macroscopical  quantities  only  in  the  rare  cases  of  j 
lipuria  and  chyluria. 

Microscopically,   fat  appears   in  the   form  of  highly 


URINE  223 

refractive  drops  and  granules  with  sharply  defined,  dark 
margins,  either  floating  free  in  the  liquid,  or  lying  upon 
other  solid  elements,  as,  for  example,  casts,  or  as  the  prod- 
uct of  fatty  degeneration  of  the  protoplasm  lying  within 
the  cells.  Often  the  latter  are  so  filled  with  fat  globules 
that  the  nucleus  is  invisible,  and  the  cell  resembles  a 
colostrum  corpuscle  (fat-granule  cells — Fig.  32). 


FIG.  32.— a,  Fatty-acid  needles;  6,  fatty  degenerated  renal 
epithelial  cells  (fat-granule  cells) ;  c,  renal  epithelial  cells ; 
d,  hyaline  cast ;  e,  cast  covered  with  renal  epithelial  cells. 

Fatty-acid  crystals  are  occasionally  seen  .together  with 
fat  drops.  They  appear  as  straight  or  wavy  needles, 
which  frequently  have  a  stellate  arrangement,  or  radiate 
from  a  fat  drop. 

Fat  is  stained  black  by  a  1  per  cent,  solution  of  osmic 
acid,  and  bright  red  by  a  saturated  alcoholic  solution  of 


224    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Sudan  III.     It  iacharacterized  chemically  by  its  solubility 
in  ether,  chloroform,  and  carbon  bisulphide. 

Organized  Sediments 

Epithelium. — The  epithelial  cells,  which  are  found  in 
urinary  sediment,  have  a  varied  appearance   (Fig.  33). 


FIG.  33.— Epithelial  Cells,  a,  From  the  male  urethra;  6,  from 
the  vagina ;  c,  from  the  prostate  ;  d,  from  Cowper's  glands ; 
e,  from  Littre's  glands ;  /,  from  the  female  urethra ;  g,  from 
the  bladder  (according  to  Loebisch). 

They  may  be  divided,  according  to  their  origin,  into  three 
groups : 

1.  Epithelium  from  the  urinary  passages. 

2.  Renal  epithelium. 

8.  Epithelium  from  the  genitalia  (prepuce,  vagina, 
vulva) . 

Careful  histological  investigations  have  shown  that  the 
entire  urinary  tract,  from  the  pelvis  of  the  kidney  to  the 


URINE  225 

fossa  navicularis  urethrae,  is  lined  with  stratified  epithe- 
lium, which,  with  the  exception  of  slight  local  differences, 
is  of  the  same  type.  The  superficial  layer  usually  consists 
of  polygonal,  mono-  or  polynuclear,  flat  epithelial  cells, 
which  have  indentations  on  their  under  surface.  The 
cells  of  the  second  layer,  which  is  usually  composed  of 
several  rows  of  oval,  pear-shaped,  tailed  cells,  fit  into 
these  indentations.  The  lowest  layer  consists  of  small, 
polygonal,  or  round  cells  with  large  nuclei.  The  anterior 
portion  of  the  urethra  to  the  fossa  navicularis  is  lined  with 
stratified  squamous  epithelium,  while  the  superficial  layer 
of  the  pars  cavernosa  and  membranacea  urethra  consists, 
according  to  most  authors,  of  cylindrical  cells. 

Any  of  these  forms  of  cells  may  be  found  in  the  sedi- 
ment of  [the  urine  in  varying  quantity,  without  it  being 
possible  to  tell  from  their  appearance  from  what  portion 
of  the  urinary  tract  they  come.  Their  examination  can 
only  reveal  which  layer  of  the  epithelial  lining  is  in  the 
process  of  desquamation.  The  wide-spread  idea  that  the 
appearance  of  tailed  epithelial  cells  in  the  sediment  of  the 
urine  depends  upon  the  existence  of  pyelitis  must,  there- 
fore, be  discarded  as  erroneous,  since  histological  inves- 
tigation has  shown  that  this  form  of  cell  is  in  no  wise 
peculiar  to  the  pelvis  of  the  kidney. 

Normal  urine  always  contains  in  the  nubecula,  or  in  its 
precipitate,  occasional  'flat  epithelial  cells  (Fig.  33).  In 
inflammatory  processes  of  the  urinary  tract  numerous 
epithelial  cells  of  varying  form  appear  in  the  sediment  in 
addition  to  the  other  products  of  inflammation.  Epithe- 
lial cells  may  show  all  kinds  of  degeneration:  they  may 
be  swollen,  the  nuclei  indistinct,  the  protoplasm  filled  with 
vacuoles,  or  in  the  process  of  fatty  or  hyaline  degeneration. 
Renal  Epithelial  Cells  (Fig.  32).— These  appear  as  round 
or  cuboid,  sharply  bordered,  cells  with  large,  often  vesic- 


226    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

ular,  nuclei.  The  protoplasm  is  finely  granular,  and  usu- 
ally in  a  state  of  more  or  less  marked  fatty  degeneration. 
They  are  slightly  larger  than  leucocytes,  from  which  they 
can  often  be  distinguished  only  by  their  distinct  nucleus 
and  sharply  defined  contour.  If  they  lie  singly  they  are 
not  always  easy  to  recognize,  because  of  their  close  resem- 
blance to  the  epithelial  cells  of  the  lowest  layer  of  the  uri- 


FlG.  34.— Squamous  Epithelial  Cells. 

nary  passages.  Only  their  characteristic  arrangement  in 
epithelial  threads,  or  the  simultaneous  presence  of  casts, 
upon  which  they  frequently  lie,  identifies  them  as  renal 
cells.  In  icteric  urine  they  are  frequently  stained  yellow. 

The  presence  of  renal  epithelium  in  the  sediment  always 
indicates  disease  of  the  kidney. 

Epithelial  Cells  from  the  Genitalia  (Fig.  34)  appear  in 
the  urine  as  large  squamous  cells,  and  conie  in  the  male 


URINE  227 

from  the  prepuce;  in  the  female  from  the  vulva  and 
vagina.  These  cells  frequently  appear  folded  and  with 
curled  edges.  In  the  urine  of  women,  in  which  they  are 
present  normally  in  great  numbers,  white  flakes  are  often 
seen  with  the  naked  eye,  which  are  found  microscopically 
to  be  continuous  membranes  of  large  squamous  cells. 

Leucocytes  (Pus-corpuscles — Plate  IX,  Fig.  M). — The 
sediment  of  normal  urine  contains  a  few  isolated  leuco- 
cytes, which,  however,  have  no  diagnostic  significance. 
In  the  urine  of  women  with  leucorrhcea,  leucocytes  appear 
in  great  numbers  without  indicating  disease  of  the  urinary 
tract.  Leucocytes  appear  in  the  urine  in  great  numbers 
as  constituents  of  pus.  The  urine  then  appears  more  or 
less  turbid,  and  on  standing  a  sediment  is  formed,  the 
character  of  which  differs  with  the  reaction  of  the  urine. 
In  acid,  amphoteric,  and  faintly  alkaline  urine,  pus  forms 
a  non-transparent,  flaky,  gray,  or  yellowish-white  sedi- 
ment, which  appears  homogeneous,  or  contains  threads  or 
clumps  of  blood,  crystals,  etc.  In  contradistinction  to 
phosphatic  sediment,  which  resembles  it  in  appearance, 
purulent  sediment  is  insoluble  in  acetic  acid,  and  upon 
the  addition  of  caustic  potash  (Donne's  test)  becomes  a 
glairy,  mucoid,  stringy  mass,  which  represents  the  puru- 
lent sediment  of  strongly  alkaline  and  ammoniacal  urine. 
When  such  a  sediment  is  poured  from  the  vessel  it  fre- 
quently slides  out  as  a  'gelatinous  coagulum. 

The  microscopical  picture  which  leucocytes  present  also 
depends  upon  the  reaction  of  the  urine.  In  acid  and 
faintly  acid  urine  they  appear  as  round,  colorless  cells 
with  granular,  refractive  protoplasm.  They  have  one  or 
more  nuclei,  which  are  only  clearly  seen  after  the  addition 
of  reagents.  If  a  drop  of  acetic  acid  is  allowed  to  run 
under  the  cover-glass,  the  granulation  disappears,  the 
protoplasm  becomes  transparent,  and  one  or  more  irreg- 


228    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

ular,  often  horseshoe-shaped,  nuclei  with  nucleoli  are 
visible. 

In  strongly  alkaline  and  ammoniacal  urine  pus- 
corpuscles  are  usually  in  a  state  of  degeneration.  They 
-are  glassy,  swollen,  transparent,  and  the  granules  have 
disappeared,  or  as  a  narrow  border  surround  a  clear  central 
zone  in  which  the  nucleus  is  still  visible.  As  the  degener- 
ation advances,  the  contour  of  the  individual  cells  fades, 
the  nuclei  become  indistinct,  and  the  leucocytes  finally 
form  a  granular  detritus,  in  which  isolated  free  nuclei  and 
but  few  unaltered  cells  are  visible. 

Their  insolubility  in  acetic  acid  prevents  confusion  of 
•these  products  of  the  decomposition  of  leucocytes  with 
amorphous  phosphates. 

Red  Hood-corpuscles  appear  as  round,  biconcave,  yel- 
low discs,  lying  singly  or  in  groups.  In  renal  hemor- 
rhage they  appear  arranged  in  cylinders  (blood-casts),  or 
lying  upon  casts.  Frequently  they  cover  the  entire  field, 
and  completely  obscure  the  other  solid  constituents.  The 
latter  can  be  seen  only  after  the  red  blood-corpuscles  have 
been  dissolved  by  allowing  a  drop  of  distilled  water  or 
dilute  acetic  acid  to  run  under  the  cover-glass. 

Erythrocytes  frequently  show  alterations  in  form  and 
color,  depending  upon  the  concentration  of  the  urine,  its 
reaction,  and  the  length  of  time  that  they  have  been  pres- 
ent in  it.  In  faintly  acid  urine  they  remain  unaltered  for 
some  time,  while  in  concentrated  and  highly  acid  urine 
they  appear  shrivelled  and  crenated.  In  the  presence  of 
a  strongly  alkaline  reaction  they  degenerate,  and  finally 
become  decomposed,  forming  clumps  and  flakes  consisting 
of  blood-pigment  (cf .  haemoglobin) . 

After  long  contact  with  the  urine,  and  in  very  dilute 
urine,  their  pigment  is  extracted,  they  swell  up,  and 
•appear  as  colorless,  annular  bodies  (shadow  corpuscles), 


URINE  229 

which  are  recognized  with  difficulty,  especially  when  they 
are  isolated. 

Red  blood-corpuscles  may  occasionally  be  confused 
with  yeast  cells.  For  differentiation,  a  drop  of  1  to  2  per 
cent,  acetic  acid  is  added ;  red  blood-corpuscles  are  almost 
completely  dissolved  and  become  invisible,  while  yeast 
cells  remain  unaltered. 

Frequently  in  bloody  urine  clots  are  found,  which  can 
be  detected  with  the  naked  eye.  They  differ  widely  in 
their  macroscopical  appearance ;  they  appear  sometimes  as 
irregular  clumps  or  flakes,  sometimes  as  thready,  rod- 
shaped,  or  vermiform  objects,  which  may  be  as  thick  as  a 
ringer  and  several  centimetres  in  length.  They  may  be 
red,  reddish-brown,  or  blackish-brown,  or  frequently 
grayish-white.  The  latter  is  true  of  coagula  which  have 
been  in  the  urine  a  long  time. 

The  long,  slim  clots  have  diagnostic  significance.  Since 
they  are  thought  to  be  formed  in  the  ureter,  their  appear- 
ance suggests  that  the  seat  of  the  hemorrhage  is  in  the 
ureter  itself,  in  the  kidney,  or  its  pelvis.  The  possibility 
that  such  coagula  may  owe  their .  form  to  their  passage 
through  the  urethra  must,  however,  be  considered.  The 
form  of  clot,  therefore,  does  not  suffice  to  determine  the 
location  of  the  hemorrhage;  on  the  contrary,  all  the  ac- 
companying symptoms  of  the  hsematuria  must  be  con- 
sidered. 

Microscopically,  blood-coagula  appear  as  a  net-work 
of  fibrin,  whose  meshes  are  filled  with  a  varying  number 
of  unaltered  and  altered  blood-corpuscles.  Blood  is  de- 
tected microchemically  by  the  tests  described  on  p.  66. 

Fibrin  (Plate  IX,  Fig.  N). — In  addition  to  the  above- 
described  blood-clots,  whose  framework  is  composed  of 
fibrin,  structures  composed  entirely  of  fibrin  appear  in  the 
urine  following  hsematuria. 


230     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Macroscopical  quantities  of  fibrin  are  passed  in  the 
urine  in  the  rare  cases  of  so-called  fibrinuria  and  chyluria, 
in  which  they  form  white,  gelatinous  clots  either  before  or 
after  the  urine  is  passed. 

Microscopically,  fibrin-clots  are  found  to  be  composed 
of  bundles  of  parallel,  highly  refractive,  white,  or  reddish- 
yellow  fibres.  In  doubtful  cases  they  can  be  recognized 
by  means  of  Weigert's  fibrin  stain  (Fig.  53). 

Casts  (Plate  VIII,  Fig.  L,  and  Plate  X,  Fig.  0).- 
Casts  are  microscopical,  cylindrical  structures  of  varying 
length  and  thickness,  with  sharply  defined,  parallel  sides, 
and  rounded  ends.  They  are  sometimes  straight  and 
sometimes  wavy,  and  are  often  bent  or  indented.  Fre- 
quently one  end  of  the  cast  appears  to  have  been  broken 
off.  Fragments  are  also  often  seen,  which  can  be  recog- 
nized only  by  comparison  with  intact  casts.  Casts  are 
renal  in  origin,  and  owe  their  form  to  the  urinary  tubules, 
from  which  they  are  washed  by  the  urine. 

The  following  varieties  are  distinguished:  (1)  Casts 
composed  of  cells.  (2)  Granular  casts.  (8)  Hyaline 
casts.  (4)  Waxy  casts. 

Casts  of  the  first  group  are  designated,  according  to 
the  form  of  cell,  as  epithelial,  blood,  and  leucocyte  casts. 

The  renal  epithelial  cells,  of  which  the  epithelial  casts 
are  composed,  are  almost  never  unaltered,  being  usually 
in  a  state  of  granular  or  fatty  degeneration. 

If  the  degeneration  is  more  advanced,  the  outline  of 
the  cells  is  obliterated,  their  nuclei  are  difficult  to  recog- 
nize, or  have  entirely  disappeared,  and  finally  their 
epithelial  character  is  completely  lost,  and  the  picture  of 
.  the  granular  cast  is  produced.  Frequently  one-half  of  a 
cast  has  still  a  distinct  epithelial  appearance,  while  the 
other  appears  granular. 

Granular  casts  have  a  granular  surface,  which  gives 


URINE  231 

them  a  dark  appearance.  These  granules,  which,  depend- 
ing upon  their  origin,  may  consist  of  albumin  or  fat,  are 
sometimes  small  and  sometimes  large,  so  that  a  distinc- 
tion is  made  between  finely  and  coarsely  granular  casts. 
If  the  granules  consist  principally  of  minute  fat  droplets, 
the  casts  are  called  fat-granule  casts,  and  attract  notice 
by  their  glistening  appearance,  which  they  owe  to  the 
high  refractive  power  of  the  fat. 

In  the  urine  of  women  numerous  long,  granular  epi- 
thelial cells  from  the  external  genitalia  are  often  seen,  and 
make  the  recognition  of  granular  casts  more  or  less  diffi- 
cult, depending  upon  the  experience  of  the  observer;  the 
usually  distinct  nucleus  of  the  epithelial  cells,  however, 
prevents  confusion. 

Hyaline  casts  have  a  pale,  homogeneous,  transparent, 
basic  substance,  whose  margin,  however,  is  always  dis- 
tinct. These  structureless  and  colorless  objects  are  fre- 
quently so  delicate  that  they  can  be  recognized  only  with 
difficulty.  Their  detection  is  simplified  by  the  deposits 
which  they  frequently  have  upon  them.  Cellular  elements, 
as  renal  epithelium,  red  and  white  blood-corpuscles,  as 
well  as  fat  drops,  granular  detritus,  micro-organisms, 
and  salts,  frequently  entirely,  or  partially,  cover  them. 

To  simplify  the  detection  of  hyaline  casts  they  may  be 
stained,  by  adding  to  the  sediment  a  few  drops  of  LugoVs 
solution,  or  a  saturated  watery  solution  of  picric  acid. 
Thin,  watery  fuchsin  or  methylene-blue  solutions  may 
also  be  ussd  for  this  purpose. 

Waxy  casts  have,  like  hyaline,  a  homogeneous  basic 
substance,  but  are  broader,  larger,  and  of  tougher  consist- 
ency. 

They  are  waxy,  moderately  refractive  in  appearance, 
yellow  in  color,  and  frequently  show  deep  indentations; 
occasionally  very  broad,  short  forms  are  seen. 


232    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Cylindroids  (Fig.  35) ,  which  are  found  both  in  normal 
and  pathological  urine,  must  be  distinguished  from  true 
casts.  They  are  most  easily  confused  with  hyaline  casts. 
In  contradistinction  to  the  latter,  their  basic  substance  is 
not  homogeneous,  but  usually  shows  a  distinct  longitudi- 


FIG.  35. — a,  Cylindroids;  6,  crystals  of  calcium  oxalate; 
c,  leucocytes. 

nal  striation.  In  addition  they  have,  as  a  rule,  frayed  or 
forked  ends. 

Clumps  of  bacteria,  which  resemble  granular  casts  in 
form,  are  occasionally  found  in  the  sediment,  and  are  called 
bacterial  casts.  Examination  with  the  high  power  and 
staining  with  a  dilute  watery  solution  of  f  uchsin  or  methy- 
lene  blue  will  identify  these  structures. 

Fragments  of  Tissue. — Fragments  of  tissue  are  rarely 
found  in  the  urine.  They  may  be  easily  overlooked  in 


URINE  233 

turbid  urine,  particularly  when  it  contains  blood  or  pus. 
To  prevent  this,  such  urine  should  be  poured  into  a  flat 
dish,  in  which  it  can  be  conveniently  examined.  The 
fragments  are  removed  and  examined  separately.  Frag- 
ments of  tissue  are  passed  in  the  urine  in  tumors  of  the 


FIG.  36.  — a,  Urinary  filament,  composed  of  pus-corpuscles  and 
epithelial  cells ;  6,  urinary  filament,  composed  of  sperma- 
tozoa and  occasional  leucocytes ;  c,  urinary  filament  com- 
posed of  pus-corpuscles. 

kidney  and  urinary  passages,  in  severe  septic  cystitis, 
which  has  caused  gangrene  of  the  mucosa  of  the  bladder, 
as  well  as  in  pyelitis.  When  tumors  of  the  neighboring 
organs  have  extended  into  the  urinary  tract,  particles  of 
them  may,  of  course,  be  passed  in  the  urine.  Fragments 
of  tissue  must  be  especially  examined  histologically. 

Urinary  Filaments  (Urethral  Threads)   (Fig.   36).-— By 
urinary  filaments  are  meant  small  threads  or  flakes  which 


234     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

are  passed  in  the  urine  as  products  of  the  purulent  or 
mucoid  secretion  of  the  urethra  and  genital  glands.  They 
are  of  varying  size,  often  1  to  2  centimetres  in  length,  and 
appear  muco-gelatinous,  or  yellow  and  non-transparent, 
but  all  stages  between  these  two  types  are  seen. 

Filaments  are  present  in  the  urine  in  chronic  gonor- 
rhoea (gonorrhoeal  threads) ,  also  in  the  urine  of  neuras- 
thenic patients  having  urethrorrhcea,  and  occasionally  in 
the  first  morning  urine  of  healthy  persons. 

The  microscopical  picture  presented  by  the  urethral 
filaments  is  the  same  in  the  last  two  cases.  They  are 
composed  of  a  homogeneous,  transparent  basic  substance, 
in  which  a  varying  quantity  of  epithelial  cells,  a  few  leuco- 
cytes, and  frequently  amorphous  and  crystalline  salts  are 
embedded. 

The  urethral  threads  in  gonorrhoea  are  composed  either 
of  thick  clumps  of  pus-corpuscles  or  of  both  epithelial 
cells  and  pus-corpuscles,  sometimes  more  of  one  and  some- 
times more  of  the  other,  or  of  epithelial  cells  alone.  In 
cases  in  which  ejaculation  of  semen  has  preceded  the  pas- 
sage of  the  urine,  as  well  as  in  the  urine  of  persons  suffer- 
ing from  spermatorrhoea,  spermatozoa  are  also  present  in 
the  filaments. 

Microscopical  examination  shows  that  the  macroscopi- 
cal  appearance  of  urethral  filaments  depends  upon  their 
richness  in  cells.  The  fewer  the  cells  contained  the  nearer 
they  approach  the  type  of  the  muco-gelatinous  filaments. 

For  microscopical  examination  of  the  filaments  the  first 
morning  urine  is  best  used,  from  which  only  the  first  10 
to  15  cc  are  collected,  since  the  filaments,  particularly  the 
yellow,  are  usually  very  fragile,  and  are  easily  dissolved 
by  a  large  quantity  of  urine.  They  are  removed  with 
a  pipette  or  a  bent  needle,  and  carefully  spread  on  a 
slide. 


URINE  235 

Secretion  from  the  Genital  Glands  (Plate  X,  Fig.  P).— 
Spermatozoa  are  frequently  present  in  the  sediment  of  the 
urine.  They  are  present  in  the  urine  following  coitus  and 
pollutions,  in  diseases  of  the  genital  organs,  as  well  as 
following  convulsions,  and  in  severe  febrile  diseases,  par- 
ticularly typhoid  fever.  They  appear  sometimes  singly, 
sometimes  in  great  quantity,  and  frequently  arranged  in 
filaments.  Spermatozoa  may  also  be  found  in  the  urine 
of  women  passed  after  coitus. 

Occasionally  they  are  still  lively,  but  very  frequently 
motionless.  Large  round  cells  with  distinct  nuclei  are 
also  sometimes  seen  enclosing  spermatozoa.  Delicate, 
pale  cylindrical  objects,  with  a  homogeneous  basic  sub- 
stance, are  often  seen.  These,  the  so-called  testicular 
casts,  come  from  the  tubules  of  the  testicle,  and  resemble 
hyaline  casts.  They  are  distinguished  from  true  hyaline 
casts  by  the  simultaneous  presence  of  spermatozoa,  which 
frequently  lie  upon  them. 

Prostatic  Secretion  is  mixed  with  the  urine  in  diseases 
of  the  prostate  and  following  its  massage.  Numerous 
small,  glistening  granules,  called  lecithin  granules,  are 
then  also  present  in  the  sediment,  and  in  addition  round 
or  angular  objects  with  a  distinct  concentric  striation, 
which  are  called  prostatic  bodies,  or,  since  they  resemble 
starch  granules,  Corpora  amylacea. 

Animal  Parasites. — Of  the  animal  parasites  which  may 
appear  in  the  urine  the  echinococcus  is  of  special  interest, 
since  the  others  either  are  not  observed  in  our  latitude  or 
are  merely  present  accidentally,  and  have  no  pathogno- 
monic  significance. 

Portions  of  the  echinococcus  (Plate  XI,  Fig.  Q)  appear 
in  the  urine  when  the  echinococci  are  located  in  the  uri- 
nary tract,  or  when  an  echinococcus  cyst  has  ruptured  into 
it  from  the  neighboring  tissues.  Entire  cysts  which  may 


236    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

be  passed  in  great  numbers  are  then  found,  as  well  as  the 
characteristic  booklets  and  shreds  of  membrane,  which 
can  be  easily  recognized  by  their  distinct  stratification. 

The  following  parasites  are  more  rarely  found:  Em- 
bryos of  the  Filaria  sanguinis    (in  tropical  chyluria), 


FlG.  37.— a.  Vegetable  cells ;  b,  starch  granules ;  c,  air-bubbles ; 
d,  vegetable  fibres. 

eggs  of  the  Eustrongylus  gigas,  eggs  of  the  Distoma 
hcematobium  (in  bilharziosis) . 

The  infusoria,  Cercomonas  urinarius,  and  Tricho- 
monas  vaginalis,  which  occasionally  appear  in  the  sedi- 
ment of  the  urine  have  no  significance.  Occasionally 
amoebae,  the  Iarva3  of  flies,  and  pediculi  pubis,  are  found 
in  the  sediment  of  the  urine  as  accidental  constituents. 

Substances  Found  in  the  Sediment  Due  to  Contamination  of 
the  Urine  (Fig.  87) . — The  presence  in  the  sediment  of  food 


URINE  237 

particles,  vegetable  cells,  muscle  fibres,  etc.,  indicates  the 
contamination  of  the  urine  with  faeces.  If  this  contamina- 
tion is  due  to  a  recto-vesical  fistula,  the  urine  shows 
simultaneously  evidences  of  severe  cystitis.  Constituents 
of  the  faeces  found  in  the  sediment  are  usually,  however, 
due  to  contaminated  urinary  receptacles. 

Sputum,  hair,  vegetable  and  animal  fibres,  starch  gran- 
ules, fat,  and  fungi  may  also  be  adventitious  constituents 
of  the  urinary  sediment. 

VIII.  Bacteriological  Examination  of  the  Urine 

Collection  of  the  Urine  for  Examination 

The  urine  is  best  collected  for  bacteriological  examina- 
tion by  means  of  a  sterile  catheter,  following  a  thorough 
cleansing  of  the  external  genitalia  and  irrigation  of  the 
anterior  urethra,  which  normally  possesses  a  luxuriant 
bacterial  flora.  The  urine  first  passed,  which,  in  spite  of 
the  irrigation,  may  contain  micro-organisms  or  secretions, 
which  have  been  carried  by  the  catheter  from  the  urethra 
into  the  bladder,  is  allowed  to  escape,  and  the  following 
portion  collected  in  a  sterile  receptacle.  If  for  any  reason 
the  urine  cannot  be  obtained  per  catheter,  the  external 
genitalia  are  cleansed,  the  urethra  irrigated,  and  the  first 
portion  of  the  urine  allowed  to  escape,  which  cleanses  the 
urethra  still  further,  and  the  second  portion  used  for  ex- 
amination. The  urine  should  be  examined  as  soon  as 
possible  after  its  evacuation,  since  the  micro-organisms 
present  usually  multiply  very  rapidly. 

Preparation  of  the  Urine  for  Examination 

In  most  cases  it  is  advisable  to  centrifugalize  the  urine 
in  sterile  tubes,  and  to  use  the  sediment  so  obtained  for 
examination.  When,  however,  the  urine  is  very  rich  in. 


238    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

bacteria,  which  can  be  determined  by  examining  a  hang- 
ing drop,  it  is  sufficient  to  take  a  drop  of  it  for  examina- 
tion, as,  for  example,  in  the  so-called  bacteriuria.  Occa- 
sionally, in  the  latter  case,  no  sediment  is  obtained  by 
centrifugalization.  To  obtain  a  sediment  from  such  urine, 
absolute  alcohol  may  be  added,  which  lowers  the  specific 
gravity  of  the  liquid,  with  the  result  that,  when  centri- 
fugalized,  the  solid  constituents  are  precipitated.  To 
obtain  a  sediment  in  ammoniacal  urine,  it  is  often  neces- 
sary to  heat  it  in  a  water-bath  with  dilute  potassium 
hydrate.  In  the  two  latter  cases  the  precipitate  is,  of 
course,  unfit  for  cultural  use.  In  urine  rich  in  urates  the 
salts  are  first  dissolved  by  slight  heating;  for  this  purpose 
the  urine  may  be  placed  for  a  few  minutes  in  an  incubator 
at  37°  C. 

Method  of  Examination 

Urine  is  examined  bacteriologically  by  means  of  stained 
smears,  cultural  procedures,  and  animal  inoculation. 

Smears  are  made  in  the  usual  manner.  In  the  pres- 
ence of  a  large  amount  of  crystalline  salts  they  are,  how- 
ever, best  fixed  in  absolute  alcohol  (ten  minutes)  ;  in  the 
presence  of  fat  or  blood  in  alcohol  and  ether  (three  min- 
utes) .  The  smears  are  stained  with  dilute  borax  methy- 
lene  blue  (1:9)  two  minutes,  without  heating,  according 
to  Gram,  and  according  to  one  of  the  methods  for  detect- 
ing tubercle  bacilli. 

Cultural  Procedures. — Agar  is  best  used  for  isolating 
the  bacteria  which  appear  in  the  urine.  Special  culture 
media  are,  however,  necessary  for  the  detection  of  tubercle 
bacilli  and  gonococci. 

Animal  inoculation  is  used,  as  a  rule,  only  in  the  diag- 
nosis of  tuberculosis.  Guinea-pigs  are  used  as  test- 
animals,  and  are  inoculated  with  the  sediment  obtained 


URINE  239 

by  centrifugalizing  the  urine  in  the   manner   described 
under  examination  of  the  sputum. 

The  pathogenic  bacteria  of  importance  in  examination 
of  the  urine  are  the  bacilli  belonging  to  the  group  of  B. 
coli,  tubercle  bacilli,  staphylo-,  strepto-,  and  gonococci,' 
typhoid  bacilli,  Proteus  vulgaris,  and  B.  pyocyaneus. 

Frequently  a  mixture  of  different  organisms  is  seen  in 
the  stained  smears.  It  is  then  impossible  to  tell  which 
bacteria  should  be  considered  as  the  exciting  cause  of  the 
disease.  ^  Frequently  the  picture  which  the  bacterial  flora 
presents  in  these  cases  is  not  constant,  but  varies  with  the 
different  examinations.  Such  a  condition  is  caused  by 
the  bacteria  of  decomposition,  which  have  become  secon- 
darily located  in  the  diseased  bladder,  and  therefore  the 
isolation  and  identification  of  the  various  bacteria  have 
merely  a  scientific,  and  no  diagnostic,  value 

Bacterium  Coli  (Plate  XI,  Fig.  R)  is  by  far  the  most 
frequent  exciting  cause  of  cystitis  and  pyelitis ;  bacteriuria 
caused  by  it  is  often  observed.  The  urine  is  acid  in  re- 
action so  long  as  none  of  the  bacteria  of  decomposition 
have  gained  entrance  to  the  bladder.  Under  the  name  of 
B.  coli  is  included  a  group  of  bacilli  whose  type  is  the  B 
coli,  cultivated  by  Eschericli  from  the  intestine  of  the 
nursing  child.  The  different  members  of  this  group  vary 
in  their  morphological  and  biological  characteristics,  de- 
pending, to  a  certain  extent,  upon  external  conditions ;  but 
they  also  possess  a  number  of  constant  characteristics., 
Among  the  latter  are  their  luxuriant  growth  upon  all  the 
usual  culture  media,  their  very  slight  tendency  to  liquefy 
gelatine  and  to  form  spores,  and  the  fact  that  they  are 
decolorized  by  Gram.  The  different  members  vary  in 
their  ability  to  ferment  sugar,  coagulate  milk,  form  in- 
dol,  etc. 

B.  coli  appears  in  the  stained  smear  made  from  uri- 


240    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

nary  sediment  as  a  plump,  straight  rod  with  rounded  ends, 
and  of  varying  length.  The  bacteria  lie  singly  in  pairs 
or  in  groups  and  frequently  form  chains ;  more  rarely  they 
lie  within  the  cells.  They  are  decolorized  in  specimens 
stained  according  to  Gram. 

They  are  easily  cultivated  on  agar.  After  twenty- four 
hours'  growth  at  37°  C.  grayish-white  colonies  have  devel- 
oped. Concerning  the  identification  of  the  cultivated 
bacteria  by  means  of  transplantation  on  litmus-whey, 
neutral-red  agar,  milk,  etc.,  cf.  the  Examination  of  the 
Faeces  for  Typhoid  Bacilli  (p.  108) . 

Staphylococci  and  Streptococci  appear  as  independent 
exciters  of  disease  more  rarely  than  B.  coUj  but  they 
appear  frequently  as  producers  of  mixed  infection  in  cys- 
titis and  pyelitis. 

Both  varieties  of  cocci  stain  according  to  Gram,  and 
are,  therefore,  especially  conspicuous  in  smears  so  stained. 
Staphylococci  frequently  lie  within  the  cells.  For  differ- 
ential diagnosis  only  gonococci  come  into  consideration, 
from  which  Staphylococci  and  streptococci  are  easily  dis- 
tinguished by  their  form,  staining  characteristics,  and 
the  ease  with  which  they  can  be  cultivated  upon  the  usual 
culture  media.  In  regard  to  their  cultural  characteristics, 
cf.  Examination  of  Sputum. 

Tubercle  Bacilli  (Plate  XII,  Fig.  S).— Urine  passed  in 
tuberculosis  of  the  urinary  tract  is  acid  in  reaction  so  long 
as  the  bacteria  of  decomposition  have  not  gained  entrance 
to  the  bladder.  Acid  purulent  urine,  in  which  no  bacteria 
are  detected  either  in  stained  smears  or  by  means  of  cul- 
tural procedures,  always  arouses  suspicion  of  tuberculosis. 

Specimens  are  stained  for  tubercle  bacilli  in  the  same 
manner  as  in  the  examination  of  the  sputum.  Tubercle 
bacilli  in  the  urine  do  not  differ  in  appearance  from  the 
picture  which  they  present  in  the  sputum. 


URINE  241 

The  quantity  in  which  they  appear  in  the  urine  varies 
greatly.  In  tubercular  cystitis  they  are  often  present  in 
great  numbers,  lying  either  singly  or  in  groups,  and  fre- 
quently in  characteristically  plaited  or  S-shaped  arrange- 
ment. In  other  cases,  especially  in  tuberculosis  of  the 
kidney,  their  detection  is  extremely  difficult,  and  a  great 
number  of  specimens  must  be  examined  before  the  first 
bacillus  is  found.  When  the  attempt  to  detect  the  bacilli 
in  the  sediment  of  urine,  centrifugalized  in  the  usual 
manner,  fails,  it  may  occasionally  succeed  if  as  large  a 
quantity  of  urine  as  possible  is  allowed  to  stand  about 
twelve  hours  in  a  conical  glass  (containing  a  small  piece 
of  thymol),  and  the  lowest  portion  withdrawn  and  centri- 
fugalized. If  no  precipitate  is  formed  by  sedimentation, 
as  large  a  quantity  of  urine  as  possible  is  centrifugalized 
in  one  and  the  same  tube. 

Cultural  methods  usually  fail  in  the  examination  of 
the  urine  for  tubercle  bacilli,  since  their  cultivation,  even 
upon  Hesse's  agar,  succeeds  only  when  they  are  present  in 
great  numbers. 

Animal  inoculation  yields  more  certain  results,  since 
it  may  be  positive  even  when  no  tubercle  bacilli  can  be  de- 
tected in  the  stained  smears.  In  the  examination  of  the 
urine  for  tubercle  bacilli  the  frequent  presence  of  smegma 
bacilli  must  be  borne  in  mind.  Both  belong  to  the  group 
of  acid-fast  bacilli,  and'cannot,  therefore,  be  distinguished 
from  one  another  in  stained  smears.  Neither  do  the  stain- 
ing methods,  suggested  by  CzapUwski,  Pappenheim,  and 
others  (cf.  p.  331),  by  which  only  the  tubercle  bacilli  are 
stained  red,  while  the  smegma  bacilli  are  stained  blue, 
allow  a  differential  diagnosis  to  be  made  with  certainty! 
These  methods  are,  of  course,  especially  inadaptable  when 
only  isolated,  suspicious-looking  bacilli  are  detected  in 
the  smears.  Cultural  methods  are  also  of  no  service  in 


242     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

differential  diagnosis,  since  the  cultivation  of  smegma 
bacilli  is  as  yet  impossible,  and  the  cultivation  of  tubercle 
bacilli  from  the  urine  does  not  always  succeed.  Animal 
inoculation  alone  can  furnish  the  proper  means  of  differen- 
tiation, since  smegma  bacilli  are  not  infectious  for  guinea- 
pigs.  Animal  inoculation  should,  therefore,  be  used  in 
every  case  in  which  acid-fast  bacilli  are  detected  in  urine 
obtained  in  accordance  with  the  above-mentioned,  pre- 
cautions. 

Procedure  of  the  Animal  Test 

For  the  animal  test,  for  which  guinea-pigs  (half  grown, 
about  250  gr  in  weight)  are  taken,  the  thoroughly  centri- 
fugalized  urinary  sediment  is  used  after  treating  same  in 
physiological  salt  solution  or  in  bouillon.  The  inocula- 
tion is  made  subcutaneously  in  the  inguinal  region  after 
shaving  and  cleansing  with  alcohol.  The  preference  is 
given  to  the  subcutaneous  injection  against  the  intra- 
peritoneal,  because  with  other  pathogenic  germs  present 
the  animals  often  die  after  the  intra-peritoneal  injection 
much  sooner  from  peritonitis  and  sepsis.  Furthermore, 
the  onset  of  the  disease  after  subcutaneous  inoculation  can 
be  observed,  because  it  always  starts  at  first  with  a  local- 
ized tuberculosis  on  the  point  of  inoculation,  which  also 
goes  to  show,  that  the  infection  was  brought  on  by  the 
injected  material. 

If  the  test  proves  to  be  positive,  a  swelling  of  the 
regional  glands  (popliteal  glands)  sets  in  during  the 
second  or  the  beginning  of  the  third  week;  sometimes  also 
an  infiltration  is  forming  on  the  point  of  inoculation, 
afterward  the  tuberculosis  invades  the  inner  organs.  If 
these  enlarged  glands  are  removed— provided  they  are 
tubercular— smears  taken  from  the  glandular  juice  will 
show  the  tubercle-bacilli,  and  the  diagnosis  of  tubercu- 


URINE  243 

losis  can  be  made  as  early  as  in  the  second  or  third  week 
after  the  inoculation.  It  is  not  necessary  to  make  sections 
in  order  to  find  the  tubercle  bacilli.  The  extirpation  of 
the  glands  is  borne  well  by  the  animals  without  influen- 
cing the  progress  of  the  tuberculosis.  Bloch  has  recom- 
mended to  squeeze  the  inguinal  lymph-glands  before  inoc- 
ulating subcutaneously,  in  order  to  accelerate  the  tubercular 
changes  by  this  mechanical  insult  as  follows:  take  the 
inguinal  fold  between  thumb  and  index-finger  and  rub 
the  inguinal  region  repeatedly,  always  with  the  two  fingers, 
going  from  underneath  up  to  the  surface,  thus  the  inguinal 
glands  are  felt  between  the  rubbing  fingers  as  small 
nodules,  and  are  squeezed  by  firm  pressing.  If  the  test 
proves  to  be  positive,  a  node  of  about  bean-size  is  found 
in  the  inguinal  region  ten  to  fourteen  days  after  the  inoc- 
ulation; if  extirpated,  it  shows  a  number  of  enlarged 
lymphatic  glands  in  inflammatory  infiltrated  tissue.  In 
the  smears  taken  from  the  glandular  juice  numerous 
tubercle  bacilli  are  found. 

The  pressing  of  the  glands  is  to  be  omitted  when 
numerous  bacteria  are  found  by  the  microscope,  because 
the  guinea-pigs,  when  squeezed,  die  sooner  from  the  in- 
fection of  these  bacteria,  while  the  animals,  when  not 
squeezed,  survive  this  infection.  Furthermore,  it  is  to 
be  noted,  that  not  seldom,  even  two  to  three  days  after  the 
inoculation,  these  squeezed  and  pressed  glands  swell  with- 
out an  existing  tuberculosis,  but  they  do  not  get  larger  in 
the  course  of  the  following  days;  sometimes  they  become 
smaller;  sometimes  abscesses  are  forming,  in  which  case 
the  pus  is  to  be  examined  for  tubercle  bacilli.  At  any 
rate,  these  glands  must  not  be  extirpated  too  soon,  it  is 
best  to  inoculate  two  animals,  but  to  squeeze  the  glands  in 
one  only. 

If  the  animals  are  killed  four  to  six  weeks  after  the 


244     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

infection,  in  dissecting,  the  inguinal  region  is  found  to 
be  infiltrated  or  ulcerating,  the  popliteal  glands  markedly 
swollen  and  a  cheesy  mass  in  the  centre ;  numerous  miliary 
nodules  in  the  spleen,  which  is  enlarged  two  to  three  times 
its  size,  and  in  the  liver;  the  peritoneal  and  bronchial 
glands  are  enlarged  with  a  cheesy  mass  in  the  centre;  in 
the  lungs  also  often  numerous  gray  nodules  are  visible. 
To  corroborate  the  diagnosis  it  is  essential  to  find  the 
tubercle-bacilli  in  the  morbid  products.  In  the  tubercular 
nodules,  which  are  rubbed  between  two  cover-glasses  for 
the  purpose  of  examination,  always  only  very  few  isolated 
bacilli  are  found. 

If  there  is  no  swelling  of  the  glands  in  the  test-animals, 
they  have  to  be  watched  for  from  six  to  eight  weeks  and 
must  then  be  killed  and  dissected. 

Typhoid  Bacilli — Cystitis  and  bacteriuria,  caused  by 
typhoid  bacilli,  have  been  frequently  described  in  recent 
years.  The  bacilli  may  be  observed  in  the  urine  as  early 
as  the  end  of  the  second  or  the  beginning  of  the  third  week 
of  the  disease,  but,  as  a  rule,  appear  later,  and  often  not 
until  convalescence.  The  urine  is  acid  in  reaction,  and 
contains  an  enormous  quantity  of  typhoid  bacilli.  These 
are,  as  a  rule,  the  only  bacteria  present. 

On  examining  the  sediment  in  a  hanging-drop,  numer- 
ous highly  motile  bacilli  are  seen.  In  stained  smears 
they  appear  as  small  rods,  which  are  decolorized  by  Gram. 
They  are  cultivated  and  identified  according  to  the  method 
described  under  Examination  of  the  Faeces. 

Gonococci. — Pure  gonorrhoeal  cystitis  is  very  rare. 
Cystitis  following  gonorrhoea  is  usually  due  to  mixed  in- 
fection. The  diagnosis  of  gonorrhceal  cystitis  is  espe- 
cially difficult,  since,  even  when  gonococci  appear  in  the 
urine  in  great  quantities,  the  possibility  that  pus  from  the 
posterior  urethra  has  become  mixed  with  the  contents  of 


URINE  245 

the  bladder  cannot  be  excluded.  Concerning  the  detec- 
tion of  gonococci,  cf.  Examination  of  the  Urethra] 
Secretion. 

Proteus  Vulgaris. — In  cystitis  excited  by  the  Proteus 
vulgaris  the  urine  is  ammoniacal  in  character.  Proteus 
vulgar  is  appears  alone,  or  with  other  micro-organisms, 
especially  Bacterium  coli. 

Microscopical  examination  reveals  rods  of  varying 
size,  which  frequently  form  long  spiral  threads,  and  for 
the  most  part  are  decolorized  by  Gram;  occasional  bacilli, 
however,  if  deeply  stained,  are  not  decolorized.  In  hang- 
ing-drops they  appear  highly  motile.  Their  growth  on 
gelatine  is  characteristic.  Delicate,  gray  colonies  develop, 
which  soon  sink  into  the  gelatine  and  produce  wavy 
excavations  with  a  whitish  mass  in  the  centre,  surrounded 
by  a  clear  area.  The  colonies  spread  over  the  culture 
media  by  forming  radiating  branches,  which  may  separate 
entirely  from  the  mother-colony.  Proteus  vulgaris  fer- 
ments grape-  and  cane-sugar,  but  not  milk-sugar,  and 
forms  a  large  amount  of  indol.  It  decomposes  albuminoid 
substances  with  the  formation  of  foul-smelling  products. 

Bacillus  pyocyaneus  has  been  found  both  as  an  indepen- 
dent exciter  of  disease,  and  with  other  bacteria,  in  cystitis. 
(Concerning  its  microscopical  and  cultural  characteristics, 
cf.  p.  53.) 


CHAPTER  VIII 

EXAMINATION   OF   THE   URETHRAL  AND 
PROSTATIC   SECRETIONS 

Bacteriological  examination  of  the  urethral  secretion  is 
directed  principally  toward  the  detection  of  gonococci, 
which  are  in  the  vast  majority  of  cases  the  exciting  cause 
of  urethritis.  Non-gonorrhoeal  urethritis  is  rare.  It  is 
usually  excited  by  Bacterium  coli,  but  staphylococci, 
pseudo-diphtheria  bacilli,  and  micro-organisms  of  the 
normal  urethral  flora,  may  excite  it. 

In  acute  urethritis  in  the  male,  the  secretion  is  taken 
from  the  urethra  by  means  of  a  platinum  wire,  and  spread 
in  a  thin,  even  layer  upon  a  cover-glass  or  slide.  In  women 
the  secretion  of  the  urethra,  or  that  of  the  cervix  uteri,  is 
used  for  examination.  Vaginal  secretion  is  absolutely 
unfit  for  use,  since  it  usually  contains  a  large  number  of 
different  micro-organisms,  among  which  the  gonococci  can 
scarcely  be  detected.  In  young  girls,  however,  the  detec- 
tion of  gonococci  in  the  vaginal  secretion  is  very  easy. 

In  chronic  gonorrhoea  in  the  male,  ' '  the  morning  drop, ' ' 
or  the  filaments  which  appear  in  the  urine,  are  examined. 
The  latter  are  most  numerous  in  the  first  morning  urine. 
Since  they  are  washed  from  the  urethra  with  the  first 
stream  of  urine,  and  are  easily  dissolved  in  a  large  quan- 
tity, only  a  small  quantity  (about  the  first  20  or  80  cc) 
is  collected  for  examination.  The  filaments  are  removed 
with  a  pipette,  and  carefully  spread  upon  a  cover-glass  or 
slide. 

246 


URETHRAL   AND    PROSTATIC   SECRETIONS       247 

Microscopical  Examination. — Smears  are  stained  ac- 
cording to  Gram,  and  with  a  very  dilute  methylene-blue  so- 
lution (Plate  XII,  Fig.  T),  which  only  slightly  stains  the 
nucleus,  but  stains  the  cocci  intensely.  The  numerous 
double-staining  methods  which  have  been  suggested  have 
no  diagnostic  value,  though  they  simplify  the  detection  of 
isolated  cocci.  The  method  of  May  and  Gruenwald  is  to 
be  recommended.  The  methods  of  Scliaeffer,  Pick,  and 
Jakobsohn,  and  Krystallowicz's  modification  of  Pappen- 
heim's  method,  should  be  mentioned.  With  the  latter, 
the  gonococci  are  stained  brilliant  red,  the  nuclei  pale 
green,  and  the  protoplasm  faintly  pink  (cf.  p.  834). 
These  methods  afford  good  results  only  when  the  smears 
are  thinly  and  evenly  spread.  The  gonococci  appear  in 
stained  smears  as  diplococci,  which  are  usually  biscuit-  or 
coffee-bean-shaped.  They  rarely  lie  singly,  but  usually  in 
groups.  In  purulent  secretion  they  lie  almost  exclusively 
within  the  pus-corpuscles,  which  often  appear  stuffed  with 
them.  In  the  first  stage  of  gonorrhoea,  in  which  the 
mucous  secretion  contains  numerous  epithelial  cells  and 
fewer  leucocytes,  the  gonococci  frequently  lie  outside  of 
the  cells,  often  almost  completely  covering  them.  They 
also  lie  for  the  most  part  outside  of  the  cells  in  the  muco- 
purulent  secretion  of  chronic  gonorrhoea.  ,  The  fact  that 
they  are  decolorized  by  Gram  is  of  value  in  differential 
diagnosis. 

Cultural  Procedures. — Gonococci  do  not  grow  upon 
the  usual  culture  media.  A  culture  medium  containing 
serum  (human  serum  is  best)  is  necessary  for  their  culti- 
vation. The  medium  must  be  prepared  in  such  a  manner 
that  the  albumin  contained  in  the  serum  is  not  coagulated. 

Wertheim^s  serum-agar,  which  consists  of  a  mixture  of 
2  to  8  parts  nutrient-agar  with  1  part  human  blood-serum, 
is  the  most  favorable  medium  upon  which  to  cultivate  them. 


248    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

In  place  of  blood-serum  other  human  serous  fluids,  as 
hydrocele-cystic,  ascites-,  and  hydrothorax-fluids  may  be 
used.  The  latter  media  are,  however,  not  absolutely  reli- 
able, since,  for  reasons  unknown,  the  gonococci  occasion- 
ally fail  to  develop.  The  serum  is  never  mixed  with  the 
agar  until  shortly  before  use,  when  it  is  heated  to  40°  C. , 
and  poured  into  the  agar,  which  has  been  melted  and  cooled 
to  50°  C. ;  the  medium  is  allowed  to  solidify  in  obliquely 
placed  tubes.  Wassermann' s  swine-serum  nutrose-agar 
should  also  be  mentioned,  upon  which,  however,  the  gon- 
ococci develop  irregularly  and  sparingly.  Concerning  the 
preparation  of  the  culture  media,  cf.  pp.  354,  855. 

Appearance  of  the  Cultures. — After  twenty- four  hours' 
growth  at  86°  C.,  round,  slightly  gray,  transparent  colo- 
nies, of  characteristic  mucoid  consistency,  and  of  about 
the  size  of  a  small  pin's  head,  have  developed.  The  in- 
dividual colonies  do  not  coalesce,  and  resemble  those  of 
streptococci.  The  numerous  degeneration  forms,  which 
may  be  seen  beside  the  typical  diplococci  in  smears  made 
even  from  twenty-four  hours'  cultures,  are  characteristic. 
The  degeneration  forms  appear  swollen,  and  stain  poorly. 

Differential  Diagnosis. — In  examining  secretions  from 
the  genital  organs,  the  morphological  and  staining  charac- 
teristics furnish  sufficient  evidence  upon  which  to  make  a 
certain  diagnosis.  The  peculiar  form  of  the  gonococci, 
their  characteristic  position,  and  the  fact  that  they  decol- 
orize by  Gram,  usually  render  it  possible  to  differentiate 
them  at  once  from  other  pyogenic  cocci.  Occasionally  the 
detection  of  suspicious-looking  diplococci  may  necessitate 
cultural  procedures — namely,  when  the  diagnosis  is  of 
great  importance  (marriage  consent,  medico-legal  cases) . 
In  such  cases,  in  addition  to  the  serum  cultures,  smears 
are  made  upon  ordinary  agar,  since  the  absence  of  growth 
upon  the  latter  is  of  especial  diagnostic  value.  In  cases 


URETHRAL  AND   PROSTATIC   SECRETIONS      249 

of  chronic  urethritis  in  which  no  gonococci  are  detected 
microscopically,  cultural  procedures  usually  fail  also. 
Examination  is  therefore,  as  a  rule,  limited  to  thorough 
microscopical  examination.  This  should  be  repeated  as 
often  as  possible,  and,  if  necessary,  may  be  preceded  by 
provocative  irritation.  Cultural  methods  are  indispen- 
sable for  the  identification  of  gonococci  in  extra-genital 
diseases. 

It  is  occasionally  necessary  to  restain,  according  to 
Gram,  a  smear  which  contains  suspicious-looking  cocci 
(for  example,  in  the  examination  of  filaments) .  This  is 
done  by  removing  the  Canada  balsam  and  cedar  oil  with 
xylol,  which  is  in  its  turn  removed  with  absolute  alcohol, 
washing  with  water,  decolorizing  with  8  per  cent,  hydro- 
chloric acid  alcohol,  and  again  washing,  after  which  the 
smear  may  be  stained  according  to  Gram. 

Prostatic  Secretion 

Prostatic  secretion  is  obtained  for  examination  by 
massage  of  the  prostate  following  irrigation  of  the  anterior 
urethra.  It  is  examined  in  the  same  manner  as  the  ure- 
thral  secretion. 


CHAPTER  IX 
EXAMINATION   OF  THE   BLOOD 

I.  Determination  of  the  Specific  Gravity 

Hammerschlag's  Method. — A  mixture  of  chloroform 
(specific  gravity,  1.527)  and  benzol  (specific  gravity, 
0.880),  in  the  ratio  of  2  to  5.5,  is  placed  in  a  dry  100  cc 
glass  cylinder.  The  mixture  should  have  a  specific  grav- 
ity between  1.050  and  1.055,  and  should  fill  the  cyl- 
inder about  three-quarters  full.  A  medium-sized  drop  of 
blood  is  taken  from  the  ball  of  the  finger,  or  the  lobe  of  the 
ear,  into  the  mixture.  During  the  introduction  of  the  drop 
and  the  following  manipulations,  care  must  be  taken  that 
it  is  not  broken  up  into  smaller  drops.  If  the  drop  of 
blood  sinks  to  the  bottom,  the  specific  gravity  of  the  mix- 
ture is  lower  than  that  of  the  blood,  in  which  case  a  few 
drops  of  chloroform  are  added,  and  mixed  with  the  fluid, 
by  carefully  tipping  the  cylinder,  which  is  closed  with  the 
palm  of  the  hand.  If  the  drop  now  remains  at  the  sur- 
face, a  drop  of  benzol  must  be  added,  and  the  fluid  again 
mixed.  The  addition  of  chloroform  or  benzol  is  continued 
until  the  drop  assumes  a  fixed  position  in  the  fluid, 
neither  rising  nor  sinking.  The  benzol-chloroform  mix- 
ture and  the  drop  of  blood  are  then  of  the  same  specific 
gravity.  The  specific  gravity  of  the  mixture  is  determined 
by  means  of  an  areometer.  The  benzol-chloroform  mix- 
ture may  be  filtered  through  a  dry  filter  and  used  for 
subsequent  examinations.  The  specific  gravity  of  normal 
blood  is  1.055  to  1.060. 

250 


BLOOD  251 

II.  Determination  of  the  Freezing-Point 

The  determination  of  the  freezing-point  is  most  con- 
veniently carried  out  with  the  blood-serum.  Since  at  least 
10  cc  of  serum  are  necessary  for  each  determination,  the 
blood  must  be  obtained  by  venesection  or  cupping.  The 
freezing-point  is  determined  in  the  same  manner  as  that 
of  the  urine  (cf .  p.  137) . 

III.  Estimation  of  Haemoglobin 

Haemoglobin  may  be  estimated  either  by  estimating 
the  intensity  of  the  color  of  the  blood  or  the  iron  contained 
in  it.  Since  a  large  quantity  of  blood  is  necessary  for  the 
exact  estimation  of  iron,  and  since  such  an  estimation 
consumes  considerable  time,  only  the  methods  for  esti- 
mating the  intensity  of  the  color  of  the  blood  are 
used  for  clinical  and  practical  purposes.  These  allow 
the  rapid  estimation  of  haemoglobin  with  a  very  small 
quantity  of  blood.  Of  the  various  forms  of  apparatus 
which  have  been  suggested  for  the  estimation  of  haemo- 
globin, the  two  following  can  be  recommended  for  clinical 
use: 

1.  SahWs  Modification  of  Gowers'  Hcemoglobinometer. 
—The  instrument  consists  of  two  glass  tubes  of  exactly 
equal  diameter,  one  of  which  is  three-quarters  full  of  a 
solution  of  ho3matin  chloride  and  closed  at  both  ends. 
The  second  is  closed  at  but  one  end,  carries  a  scale  with 
divisions  from  10  to  120,  and  receives  the  blood  to  be  ex- 
amined. Both  tubes  are  set  in  a  black  frame  which  has 
a  white  background,  so  that  differences  in  color  can  be 
easily  recognized.  In  addition,  a  capillary  pipette  for 
measuring  20  cubic  millimetres,  a  dropper  for  diluting 
the  blood,  and  a  bottle  for  dilute  hydrochloric  acid,  are 


252    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

furnished  with  the  apparatus.    The  haemoglobin  is  esti- 
mated as  follows : 

A  few  drops  (to  the  mark  ten  or  twenty)  of  a  dilute 
(0.2  per  cent.)  hydrochloric  acid  solution  are  placed  in 
the  graduated  tube.  The  capillary  pipette  is  now  filled 
with  blood  to  the  mark  by  suction,  and  the  blood  quickly 
blown  out  upon  the  bottom  of  the  tube  containing  hydro- 
chloric acid.  The  tube  is  thoroughly  shaken,  whereupon 
the  color  of  the  blood  is  altered,  owing  to  the  formation  of 
haematin  chloride.  The  solution  becomes  dark  brown, 
and  upon  dilution  with  distilled  water  assumes  the  color 
of  the  test  fluid.  The  dilution  with  distilled  water  must 
be  made  carefully,  a  drop  at  a  time.  The  level  of  the 
liquid  in  the  tube  shows  on  the  scale  the  quantity  of 
haemoglobin  present.  One  hundred  on  the  scale  represents 
the  normal  quantity  of  haemoglobin.  This  estimation  is 
best  performed  by  daylight,  and  yields  results  thoroughly 
useful  for  practical  purposes. 

2.  Tallquist's  HcemogloMn  Scale. — This  is  an  empirical 
scale  of  colors,  which  represent  the  shades  of  blood-red 
corresponding  to  definite  percentages  of  haemoglobin.  A 
book  of  filter-paper  is  furnished  with  the  scale.  The  esti- 
mation of  haemoglobin  with  this  scale  is  very  simple;  a 
drop  of  blood  from  the  ball  of  the  finger,  or  the  lobe  of 
the  ear,  is  absorbed  with  a  leaf  of  the  filter-paper.  The 
color  of  the  drop  is  then  compared  with  the  scale;  the 
number  opposite  the  shade  which  most  nearly  corresponds 
to  it  gives  the  percentage  of  haemoglobin.  This  method 
is  not  very  accurate  (errors  of  10  to  20  per  cent.),  but  is 
very  simple,  and  quickly  performed.  Grawitz  recom- 
mends that  the  blood-spot  be  cut  out  with  scissors  and 
placed  upon  the  color  scale  directly,  since  the  colors  can 
thus  be  more  accurately  compared. 


BLOOD  253 

IV.  Enumeration  of  Blood-Corpuscles 

The  red  and  white  blood-corpuscles  are  counted  with 
the  Tlioma-Zeiss  hsemocytometer.  This  consists  of  two 
mixing-pipettes  and  a  counting  chamber.  The  mixing- 
pipettes  are  capillary  tubes,  about  10  centimetres  in  length, 
and  with  a  bulb  in  their  upper  half,  which  contains  a 
freely  movable  glass  bead ;  0. 5  and  1  are  marked  on  the 
capillary  tubes  below  the  bulb,  and  101  and  11  respec- 
tively above  it.  The  mixing  pipette  with  the  mark  101 
is  used  for  counting  the  red,  that  with  the  mark  11  for 
counting  the  white  corpuscles. 

The  counting  chamber  consists  of  a  slide,  upon  which 
a  glass  frame  with  a  circular  opening  is  cemented.  In 
the  centre  of  the  opening  is  a  round  glass  plate,  upon  the 
surface  of  which  a  network  of  large  and  small  squares  is 
marked.  The  frame  extends  exactly  0. 1  millimetre  above 
the  surface  of  this  glass  plate,  so  that  if  a  cover-glass  is 
placed  upon  the  frame,  its  under  surface  is  exactly  0. 1 
millimetre  above  the  surface  of  the  glass  plate. 

The  red  blood-corpuscles  are  counted  in  the  following 
manner:  A  drop  of , blood  is  drawn  up  into  the  proper 
pipette  to  the  mark  0.5  the  excess  of  blood  removed 
with  the  tip  of  the  finger,  and  a  2  per  cent,  solution  of 
sodium  chloride  at  once  drawn  up  to  the  mark  101.  Care 
must  be  taken  that  no  air  enters  the  pipette.  The  pipette 
is  shaken  to  obtain  even  dilution  of  the  blood  in  the  bulb. 
Three  or  four  drops  are  now  blown  from  the  pipette,  and 
a  medium-sized  drop  placed  upon  the  glass  plate  of  the 
counting  chamber.  The  drop  is  covered  with  a  cover- 
glass,  care  being  taken  that  no  bubble  of  air  is  formed. 
The  cover-glass  must  be  in  such  close  contact  with  the 
frame  that  Newton's  rings  are  seen.  The  counting  cham- 
ber is  then  placed  with  its  centre  under  the  microscope 


254    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

(magnification  of  about  180  to  220) ,  so  that  the  network 
of  lines  and  the  red  blood-corpuscles  lying  upon  it  are 
clearly  seen. 

The  network  of  the  Thoma-Zeiss  apparatus  consists  of 
sixteen  large  triple-contoured  squares.  Each  large  square 
is  divided  by  single  lines  into  sixteen  small  squares.  In 
counting  the  red  blood-corpuscles,  it  is  advisable  to  centre 
a  large  square,  and  to  count  and  note  the  corpuscles  in 
each  small  square,  counting  only  the  cells  lying  within 
the  squares  and  upon  their  upper  and  left  borders.  Five 
large  squares  ( =  5  X  16  =  80  small  squares)  are  counted. 
The  number  of  corpuscles  is  calculated  as  follows : 

The  side  of  each  small  square  is  -fa  millimetre;  its  sur- 
face is,  therefore,  -^  X  -^V  =  TTRT  °f  a  square  millimetre. 
Since  the  thickness  of  the  blood  layer  is  -fa  millimetre, 
the  volume  of  blood  contained  in  one  small  square  = 
•j-J-g-  X  yV  =  TTUTF  °^  a  cubic  millimetre. 

The  number  of  blood-corpuscles  in  a  cubic  millimetre 
of  blood  is  calculated  from  the  formula 

_m.  n.  4,000 

nr  ~' 

in  which  m  =  the  number  of  red  blood-corpuscles  counted, 
n  =  the  dilution  of  the  blood,  and  q  =  the  number  of 
small  squares  counted. 

The  enumeration  of  the  leucocytes  differs  from  the 
above  as  follows : 

1.  Instead  of  the  mixing-pipette  with  the  mark  101, 
that  with  the  mark  11  is  used  (this  allows  a  dilution  of 
1:10  or  1:20). 

2.  A  dilute  solution  of  acetic  acid  (0. 3  to  0. 5  per  cent. ) 
is  used  as  diluent,  in  which  the  red  blood-corpuscles  are 
dissolved,  and  the  white  therefore  easily  recognized  and 
counted. 


BLOOD  255 

8.  Because  of  the  small  number  of  leucocytes  contained 
in  a  field,  a  large  number  of  squares  must  be  counted. 
For  this  purpose  Turk's  modification  of  the  Thoma-Zeiss 
counting  chamber  is  best  used.  It  has,  in  addition  to 
the  sixteen  large  squares,  a  number  of  squares  of  the  same 
size,  but  which  are  not  divided  into  smaller  squares.  This 
renders  it  possible  to  count  a  much  larger  number  of 
leucocytes.  At  least  100  to  150  leucocytes  should  be 
counted  in  each  enumeration. 

We  note  the  number  of  leucocytes  in  each  square.  We 
count  in  the  same  way  as  we  count  the  red  cells.  Illus- 
tration :  130  leucocytes  were  counted  in  40  large  squares. 
The  number  of  leucocytes  in  one  cubic  mm  then  are 

130.  4000.  10 
40.16 

(provided  that  the  blood  was  diluted  ten  times) . 

A  certain  amount  of  practice  and  great  care  in  carrying 
out  the  details  are  necessary,  in  order  to  obtain  an  accurate 
count  of  blood-corpuscles.  The  counting  chamber  and  the 
mixing  pipettes  must  be  absolutely  clean  and  dry.  After 
each  count  the  pipettes  should  be  cleansed,  first  with  a  1 
per  cent,  solution  of  sodium  hydrate,  second  with  water, 
third  with  alcohol,  and  finally  with  ether. 


V.  Histological  Examination 

(a)  Examination  of  Fresh  Specimens 

The  cover-glasses  and  slides  used  for  the  histological 
examination  of  the  blood  must  be  absolutely  clean,  and 
must  have  been  washed  with  alcohol  and  ether.  The 
under  surface  of  a  cover-glass  is  touched  to  a  drop  of 
blood,  as  it  issues  from  a  prick  of  the  finger  or  ear,  and 


256    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

placed  on  a  slide,  without  pressure,  and  without  allowing 
it  to  shift  after  it  has  touched  the  slide.  The  blood  spreads 
spontaneously  in  a  very  thin  layer.  If  the  specimen  has 
been  properly  prepared,  the  cells  lie  in  the  centre  detached 
from  one  another,  and  rouleaux  formation  is  seen  only  at 
the  periphery.  In  the  examination  of  the  fresh  specimen 
the  following  points  should  be  noted : 

1.  The  intensity  of  the  color  of  the  red  blood-corpuscles 
and  their  rouleaux  formation. 

2.  Morphological  alterations  in  the  red  blood-corpus- 
cles   (poikilocytosis,    presence   of   nucleated   red   blood- 
corpuscles)  . 

8.  Increase  in  the  number  and  alteration  of  the  struc- 
ture of  the  leucocytes. 

4.  Presence  of  micro-organisms  (spirilla  of  relapsing 
fever,  plasmodia  of  malaria) . 

(6)  Examination  of  Stained  Specimens 

1.  Preparation  of  Smears. — A  thin  cover-glass   (0.1  in 
thickness)  is  held  at  one  edge  in  a  pair  of  Elirlictis  blood- 
forceps,  and  another  cover-glass  is  held  in  a  pair  of  ordi- 
nary forceps,  and  the  centre  of  its  under  surface  touched 
to  a  small  drop  of  blood,  as  it  issues  from  the  finger  or 
ear.      The  cover-glass  with  the  drop  of   blood   is   then 
quickly  placed  upon  the  other,  without  pressure,  where- 
upon the  blood  spreads  in  a  capillary  layer.     The  upper 
cover-glass  is  now  seized  by  the  edge  with  the  thumb  and 
forefinger  of  the  right  hand,  and  drawn  from  the  lower. 
The  cover-glasses  are  then  set  aside  with  the  smeared 
surfaces  up.     Spectral  colors  are  seen  at  the  best  spread 
portions  of  the  specimens  when  the  glass  is  viewed  at  an 
acute  angle. 

2.  Fixation. — The  best  fixing  fluids  for  blood  specimens 
are  absolute   alcohol,    alcohol  and    ether,   and  formalin. 


BLOOD  257 

Specimens  are  fixed  in  alcohol  and  in  alcohol  and  ether 
aa,  for  five  minutes  to  twenty-four  hours.  Formalin  fixes 
in  two  to  three  minutes.  The  best  specimens  are,  how- 
ever, obtained  by  fixation  with  heat,  which,  according  to 
ElirlicWs  original  instructions,  is  carried  out  with  a  hot 
copper  plate,  at  a  temperature  of  100°  to  180°  C.  Special 
fixing-ovens  have  been  constructed  according  to  EhrlicWs 
suggestions.  Since,  however,  fixation  upon  EhrlicJi's 
copper  plate  and  in  the  special  ovens  consumes  consider- 
able time,  and  is  therefore  unsuited  for  the  daily  use  of 
the  practising  physician,  Kowarsky1  has  shortened  the 


FIG.  38. 

procedure  considerably.  The  smears  are  placed  with  the 
blood  up,  upon  a  hollow  copper  cylinder  (Fig.  88) .  A 
crystal  of  urea  is  placed  in  the  hollow  in  the  upper  surface 
of  the  cylinder.  The  cylinder  is  heated  just  above  the 
flame  of  a  Bunsen  burner,  or  alcohol  lamp,  until  the  crys- 
tal of  urea  begins  to  melt.  This  takes  place  between  182° 
to  185°  C.  The  cylinder  and  smears  are  set  aside  until 
they  have  cooled.  The  entire  fixation  takes  but  two  to 
three  minutes. 

8.  Staining. — I.    Elirlicli's     Triple    Stain. — EhrlicWs 
triacid  stain  has  the  following  composition: 


1  Dr.  A.  Kowanky,  Berliner  Wn.  Wochenschr.,  1903,  No.  10, 


I 


258     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Sat.  watery  solution  of  orange-G    .       .       .  13.0  to  14.0 
Sat.  watery  solution  of  acid  fuchsin     .       .       6.0  to  7.0 

Aqua  destillata 15.0 

Alcohol 

Sat.  watery  solution  of  methyl-green    .       .12.5 

Alcohol 10-° 

Glycerine 10-° 

The  ingredients  are  measured,  and  placed  in  the  same 
graduated  measure  in  the  above  order  and  thoroughly 
shaken.  It  is  advisable  to  filter  the  stain  before  using. 
Specimens  are  stained  five  to  ten  minutes. 

II.  Giemsa' s    Method    of  Staining.— The  azure-eosin 
stain  of   Giemsa  can  be  purchased  as  u  Giemsa  solution" 
(new) ,  and  must  be  freshly  diluted  for  the  staining  of 
blood,  so  that  one  drop  of  this  solution  is  added  for  each 
drop  of  distilled  water.     The  specimen  is  first  fixed  in 
methyl  alcohol  for  five  minutes  and  then  stained  in  the 
diluted  stain  for  fifteen  to  twenty  minutes.     The  Giemsa 
solution  shows  up  especially  the  chromatin  of  the  nuclei; 
also  parasites  of  the  blood. 

III.  Simultaneous,  Staining   and  Fixation  According 
to  May  ami   Gruemvald.—The  staining  principle  of  the 
May  and   Gruemvald  dye  is  a  chemical  combination  of 
eosin  and  methylene  blue.     If  a  0. 1  per  cent,  watery  solu- 
tion of  eosin  and  a  0. 1  per  cent,  solution  of  methylene 
blue  are  mixed,  and  allowed  to  stand  for  some  time,  a 
new  dye  is  precipitated.     This  is  collected  on  a  filter,  and 
washed  with  cold  water  until  the  water  runs  away  nearly 
colorless.     A  saturated  solution  of  the  dye  so  isolated  is 
made  with  methyl  alcohol.     This  is  placed  in  a  wide- 
mouthed  glass,  and  used  for  staining  and  fixing  blood 
specimens.     The  freshly  spread  and  air-dried   smear   is 
held  with  forceps  in  the  stain  for  two  minutes.     It  is 


BLOOD  259 

then  rinsed  in  a  beaker  of  water,  which  contains  a  few 
drops  of  the  stain,  until  it  assumes  a  pinkish-red 
color. 

IV.  Irishman's  Method  of  Staining  and  Simultaneous 
Fixation. — Leishman's  stain  can  be  purchased  ready  for 
use.  Five  to  ten  drops  of  the  stain  are  put  on  the  dry 
specimen;  after  half  a  minute 'double  the  number  of  drops 
of  aq.  dest.  are  added,  and  the  water  and  stain  are  mixed 
carefully  by  means  of  the  cover-glass.  This  staining 
mixture  remains  for  five  minutes  on  the  specimen,  after 
which  it  is  washed  off  with  water.  A  few  drops  of  water 
are  left  on  the  specimen  for  one  to  two  minutes,  until  the 
water  is  colored  a  light  green.  The  specimen  is  then 
washed,  dried  between  filter-paper,  and  then  proceeded 
with  in  the  usual  manner.  This  staining  method  gives 
excellent  results  and  is  to  be  recommended  in  the  daily 
practice. 

(c)  Sketch  of  the  Morphology  of  the  Blood 

NORMAL  BLOOD-CORPUSCLES 

Normal  Red  Blood- Corpuscles  (Normocytes).— These 
are  biconcave  discs  which  have  no  nuclei,  and  are  com- 
posed of  a  stroma  containing  haemoglobin.  Their  average 
diameter  is  7  to  7.5  /*.  With  EhrlicVs  triacid  stain, 
normal  erythrocytes  are  orange  in  color,  if  the  specimens 
have  been  properly  fixed  (at  135°  C.).  If  the  speci- 
mens have  been  fixed  at  a  lower  temperature  the  cor- 
puscles are  redder.  With  ordinary  eosin-methylene  blue 
they  are  red;  with  May  and  GruenwalcTs  stain  they  are 
pinkish-red.  In  fresh  specimens  they  are  distinctly 
yellow. 

•  Lymphocytes. — These  are  cells  about  the  size  of  red 
blood-corpuscles,  or  somewhat  larger,  with  narrow,  homo- 


260    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

geneous  (non-granular)  protoplasm  and  a  spherical  nu- 
cleus, which  nearly  fills  the  entire  cell.  With  the  tri- 
acid  stain,  the  nuclei  appear  greenish-  to  blackish-blue, 
the  protoplasm  pink;  with  eosin  and  methylene  blue  the 
nucleus  and  protoplasm  are  stained  blue.  The  lymphocytes 
constitute  about  one- fourth  of  all  the  leucocytes  normally 
present  in  the  blood. 

3.  Large  Lymphocytes. — These  differ  from  the  ordinary 
lymphocytes  only  in  size.     They  appear  in  the  blood  of 
young  children,  normally,  up  to  about  10  per  cent.,  but 
are  rare  in  healthy  adults.     They  are,  however,  commonly 
found  in  acute  and  lymphatic  leukaBmia. 

4.  Polynuclear  Neutrophilic  Leucocytes.— These  are  twice 
the  size  of  lymphocytes  and  have  several  nuclei,  or  one 
polymorphous   nucleus.       They   constitute   the  majority 
(about  75  per  cent. )  of  the  normal  leucocytes.     The  proto- 
plasm is  distinctly  granular.     The  granules  are  usually 
very  small.     With  the  triacid  stain,  the  nucleus  stains 
greenish  to  deep  blue,  the  granules  violet,  the  protoplasm 
between  the  granules  pink.     With  eosin  and  methylene 
blue  (first  eosin,  then  methylene  blue)  the  nucleus  appears 
blue,    the  granules  red,   while    the    protoplasm   remains 
colorless. 

5.  Acidophilic  or  Eosinophilic  Leucocytes-  — These  are  easy 
to  recognize,  even  in  unstained  specimens,  by  the  coarse, 
highly    refractive,    round    granules  in  their  protoplasm. 
As  the  name  implies,  the  granules  stain  with  any  acid 
stain,  and  are,  therefore,  deep  red  with  triacid  and  eosin- 
methylene  blue,  as  well  as  when  stained  according  to  May 
and  Gruemvald.      Two  varieties  of  eosinophilic  cells  are 
distinguished : 

(a)  Polynuclear  or  Normal  Eosinopliiles. — These  con- 
stitute ordinarily  2  to  4  per  cent,  of  the  total  leu- 
cocytes of  the  blood.  They  have  two  or  three  nuclei, 


BLOOD  261 

which  do  not  stain  as  intensely  as  those  of  the  neutro- 
philes. 

(b)  Mononuclear  Eosinophiles. — These  appear  in  the 
blood  only  under  pathological  conditions. 

6.  Basophilic  Leucocytes  or  Mast- Cells. — The  protoplasm 
of  these  cells  contains  coarse  granules  about  the  size  of 
eosinophilic  granules,  which  are,  however,  not  always  so 
round   nor  of  the  same  shape.      These  granules   have  a 
marked  affinity  for  basic  stains    (methylene  blue),  and 
stain,  therefore,  dark  blue  with  eosin-methylene  blue  and 
with  the  May-Gruenwald  stain.      They  are.  colorless  in 
specimens  stained  with  the  triacid  mixture. 

Mast-cells  are  mono-  or  polynuclear,  and  about  the 
size  of  the  neutrophiles,  though  sometimes  smaller.  They 
appear  in  very  small  quantity  in  normal  blood  (0.5  per 
cent.).  They  are  most  easily  detected  with  the  May- 
Gruemvald  stain. 

7.  Transition   Forms. — Their   size   corresponds  mostly 
with  the  neutrophiles.     The  protoplasma  is  mostly  baso- 
phile  and  shows  a  different  behavior;    it  appears  homo- 
geneous with  the  triacid  stain.     In  the  Giemsa  and  Leish- 
man  specimens  are  sometimes  seen  granulations,  which  at 
times  show  the  characteristics  of  neutrophile  granules  and 
at  times  the  characteristics  of  the  azurophile  granules  of 
lymphocytes.     The  nucleus  is  mostly  semicircular  or  of 
horse-shoe  shape. 

8.  Blood  Platelets. — These  are  small  (2  to  3  //  in  diam- 
eter), quadrilateral  or  round  colorless  objects,  which  fre- 
quently lie  in  groups,  and  are  present  in  great  quantity  in 
normal  blood.     They  come,  in  all  probability,  from  the  red 
blood-corpuscles,  and  are  considered  to  be  products  of  the 
decomposed  nuclear  substance.     They  are  basophilic,  and 
stain  faintly  blue  with  eosin-methylene  blue,  and  faintly 
pink  with  the  triacid  mixture. 


262    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

ABNORMAL  AND  PATHOLOGICAL  BLOOD-CORPUSCLES. 

(a)   Abnormal  and  Pathological  Red  Blood- Corpuscles 
(Plate  XIII,  Fig.  U). 

1.  Poihilocytes- — These  are  not  circular,  but  are  pear-, 
spindle-,  dumb-bell-,  or  kidney-shaped.     They  are  con- 
sidered  to   be    fragments    of    normal    erythrocytes,    and 
appear  in  the  blood  in  ansemic  conditions. 

2.  Macro-  and  Microcytes. — The  first   are   larger   than 
the  normal  erythrocytes,  and  frequently  have  no  concavity. 
They  appear  usually  in  severe  anemia.     The  microcytes 
are  smaller  than  the  normal  erythrocytes,  and  are  usually 
found  in  the  blood,  together  with  poikilocytes. 

3.  Nucleated  Red  Blood-Corpuscles  of  Normal  Size  (Nor- 
moblasts) . — The  nucleus  is  spherical,  usually  excentrically 
placed,  and  stains  very  intensely  (deep  blue) . 

4.  Large  Nucleated  Red  Blood- Corpuscles  (Megaloblasts) . 
— These  are  about  the  size  of  the  macrocytes,  and  frequently 
have  two  nuclei.  The  nucleated  erythrocytes  (both  forms) 
appear  only  in  severe  forms  of  anaemia. 

5.  Erythrocytes     with    Basophilic     Granulation. — These 
erythrocytes  have  granules  of  varying  size  (from  a  grain 
of  dust  to  one-fourth  the  size  of  the  nucleus)    in  their 
protoplasm,  which  stain  well  with  all  basic  stains,  and, 
therefore,  blue  with  all  staining  methods.     They  are  con- 
sidered by  most  authors  as  the  remains  of  nuclei,  and 
appear  in  severe  forms  of  anremia  (anaemia  following  lead- 
poisoning)  . 

6.  Polychromatophilic  Erythrocytes. — Erythrocytes  which 
have  lost  their  normal  affinity  for  acid  dyes  to  a  greater 
or  less  extent,  and  have  assumed  a  slight  affinity  for  basic 
dyes  are  designated  as  poly chromatophi lie.     They  stain  a 
bluish-red  or  violet,  instead  of  pinkish-red,  with  eosin- 


BLOOD  263 

methylene  blue.      They  do  not  appear  in  normal  blood, 
but  are  frequently  seen  in  various  forms  of  anaemia. 

(b)  Pathological  Leucocytes. 

1.  Myelocytes,  or  Mononuclear  Neutrophiles. — The  spheri- 
cal nucleus  occupies  the  greater  part  of  the  cell,  and  usu- 
ally stains  faintly — much  more  faintly  than  the  nuclei  of 
the  polynuclear  leucocytes.     The  neutrophilic  granules  of 
the  protoplasm  are,  as  a  rule,  comparatively  faintly  stained. 
These  cells  constitute  the  majority  of  the  cells  of  bone- 
marrow.     They  appear  in  great  numbers  in  the  blood  in 
myelogenic  leukaemia,  and  occasionally  in  severe  forms  of 
anaemia  in  children.     They  are  considered  to  be  the  ante- 
cedents of  the  normal  polynuclear  neutrophiles. 

2.  Eosinophilic  Marrow- Cells- — These  are   mononuclear 
eosinophiles.    They  are  usually  larger  than  the  polynuclear 
eosinophiles,  and  have  a  somewhat  smaller  nucleus  than 
the  myelocytes.     They  are  seen,  together  with  the  latter, 
in  leukaemia. 

3.  Non-Granular  Marrow- Cells. — These  are  very  large, 
delicate  cells  with  homogeneous  protoplasm.    The  nucleus 
and  protoplasm  stain  very  faintly.      The  protoplasm  is 
faintly  basophilic.     Occasionally  a  suggestion  of  neutro- 
philic granulation  is  seen  in  the  protoplasm,  so  that  these 
leucocytes  may  be   considered  as   transitional  forms  of 
myelocytes.      They  appear  in  the  blood  in  myelogenic 
leukaemia. 

VI.  Bacteriological  Examination  of  the  Blood 
1.  Examination  of  the  Blood  in  Stained  Smears 

The  blood  is  examined  in  stained  smears  in  the  diag- 
nosis of  malaria  and  relapsing  fever. 

(ft)  Malaria.— The  examination  of  the  blood  for  the 


264     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

malarial  parasite  is  best  made  with  blood  obtained  during 
the  decline  of  the  fever,  or  directly  after  it.  The  patient 
should  have  taken  no  quinine  for  several  days  before  the 
examination. 

The  blood  is  obtained  by  pricking  the  finger  or  lobe  of 
the  ear  with  a  sterile  needle.  The  edge  of  an  absolutely 
clean  cover-glass  is  touched  to  a  drop  of  blood  as  it  issues, 
and  is  placed  on  a  second  cover-glass,  or  slide,  so  that  the 
right-hand  angle  made  by  cover-glass  and  slide  is  about 
45°.  The  blood  spreads  along  the  cover-glass,  and  the 
latter  is  carried  across  the  slide  from  right  to  left.  In 
this  manner  the  blood  is  spread  in  a  thin  la*yer,  without 
pressure.  After  the  specimen  has  dried  in  the  air  it  is 
fixed  for  three  minutes  in  alcohol  and  ether  aa. 

The  staining  is  done  either  after  the  method  of  Hanson 
or  of  Giemsa. 

Manson's  Method. — Borax-methylene  blue  (cf.  p.  829), 
which  is  used  as  stock  solution,  must  be  diluted  with 
aq.  dest.  until  the  resulting  staining  solution  just  looks 
transparent  in  the  test-tube.  Into  this  diluted  staining 
solution  the  specimen  is  immersed  for  five  to  ten  seconds, 
washed  in  a  glass  of  plain  water  until  the  color  is  of  a 
dull  green,  then  dried  between  filter-paper  and  examined 
with  oil  immersion.  The  orthochromatically  stained  red 
cells  appear  green,  the  metachromatically  stained  are  gray 
blue,  the  nuclei  of  the  white  cells  and  the  parasites '  are 
blue.  The  blood  placques  are  of  a  dull  gray  blue,  whose 
margins  are  blurred  in  contradistinction  to  the  parasites 
whose  margins  are  sharply  defined.  The  pigment  of  the 
parasites  varies  from  yellow  to  dark  brown. 

Giemsa1 8  Method. — The  commercial  Giemsa  solution 
is  diluted  with  aq.  dest.  in  the  proportion  of  one  drop  of 
the  stain  to  1  cc  of  water  under  slight  agitation  in  the 
beaker.  The  specimen  is  put  into  a  small  dish,  the  speci- 


BLOOD  265 

men  being  turned  to  the  bottom,  the  diluted  staining  solu- 
tion is  poured  over  it  and  the  specimen  is  left  in  this  stain 
for  one  to  two  hours.  The  parasites  appear  blue  with  a 
refracting  red  chromatin  nucleus,  the  red  cells  are  red  and 
the  nuclei  of  the  leucocytes  are  of  lilac  or  dark  violet  color. 

Malarial  organisms  are  protozoa  which  have  a  double 
cycle  of  existence — an  asexual,  as  parasite  in  human  ery- 
throcytes,  and  a  sexual,  in  the  mosquito  (anopheles). 
They  are  divided  into  two  groups:  Large  parasites,  to 
which  the  parasites  of  tertian  and  quartan  fever  belong, 
and  the  small,  annular  parasites  of  tropical  fever  (sestivo- 
autumnal  fever).  The  types  of  fever  belonging  to  the 
different  forms  of  malaria  depend  upon  the  cycle  of  devel- 
opment of  the  parasites:  The  parasite  of  tertian  fever 
requires  forty-eight  hours  for  its  development,  that  of 
quartan  fever  seventy-two  hours,  and  that  of  tropical  fever 
twenty- four  to  forty-eight  hours.  The  infected  person  is 
free  from  fever  as  long  as  the  parasites  are  developing. 
The  fever  begins  only  after  they  have  completed  their 
development,  simultaneously  with  the  appearance  of  the 
so-called  division  forms.  Quotidian  fever  is  not  caused 
by  a  particular  parasite,  but  is  due  either  to  a  double 
tertian  or  a  triple  quartan  infection. 

The  Tertian  Parasite  (Plate  XIII,  Fig.  V,  and  Plate 
XIV,  Fig.  W) . — If  the  blood  is  obtained  during  the  height 
of  the  fever  or  during  its  fall,  and  stained  according  to 
Manson,  the  youngest  parasites  are  seen  lying  upon  the 
green  erythrocytes  in  the  shape  of  small,  blue,  ovoid 
objects,  which  appear  to  be  distinctly  annular,  as  well 
as  small  bluish  rings,  which  have  on  one  side  a  crescentic 
thickening,  and  on  the  opposite  a  slight  knob  (small  ter- 
tian parasite,  seal-ring  form).  Twenty- four  hours  later 
the  parasites  are  found  to  have  grown  considerably :  they 
are  about  twice  the  size  of  the  small  tertian  rings,  and 


266    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

some  still  show  a  distinct  annular  form  (large  tertian 
rings) ,  while  others  (large  parasites)  have  lost  the  annular 
form  and  appear  as  blue,  round,  or  irregular  discs.  Both 
the  large  tertian  rings  and  the  large  parasites  contain 
yellow  or  blackish-brown  pigment.  The  erythrocytes, 
attacked  by  the  parasites,  are  enlarged  and  pale.  At  first 
the  pigment  is  irregularly  distributed  throughout  the  pro- 
toplasm of  the  parasites.  A  few  hours  before  the  next 
paroxysm  it  is  collected  at  the  centre  of  the  parasite,  and 
the  parasite  itself  shows  a  distinct  differentiation  (divi- 
sion form).  Finally,  it  divides  into  fifteen  to  twenty- 
five  small,  round,  or  oval  blue  bodies  (spores) ,  from  which 
the  new  parasites  develop. 

In  addition  to  these  asexual  forms,  those  which  in  the 
mosquito  serve  for  the  sexual  development  of  the  parasite 
(gametes)  are  seen.  These  resemble  the  large  parasites  in 
size  and  form,  and  either  lie  free,  or  nearly  fill  the  pale, 
enlarged  erythrocytes.  They  differ  from  the  asexual  forms 
as  follows :  They  stain  less  intensely  at  the  border  (pale 
grayish-blue  or  grayish-green) ,  or  they  have  an  absolutely 
unstained  area  in  the  centre;  further,  they  show  no  differ- 
entiation in  their  protoplasm,  throughout  which  the  pig- 
ment is  irregularly  distributed.  Male  and  female  gametes, 
which  show  differences  in  staining  and  pigmentation,  are 
distinguished  in  stained  smears. 

The  Quartan  Parasite. — The  quartan  parasite  cannot 
be  distinguished  in  the  first  stage  of  its  development  from 
the  tertian.  In  specimens  obtained  during  the  decline  of 
the  fever,  rings  are  seen  which  exactly  resemble  the  small 
tertian  rings.  From  these  the  band  forms,  characteristic 
of  the  quartan  parasite,  are  developed.  The  erythrocytes 
attacked  by  the  parasites,  in  contradistinction  to  those  in 
tertian  fever,  are  neither  bleached  nor  enlarged,  and  are 
traversed  by  blue,  heavily  pigmented  bands,  which  gradu- 


BLOOD  267 

ally  enlarge,  become  quadrilateral,  and  finally  completely 
fill  the  corpuscle.  The  quartan  parasite  divides,  after 
the  pigment  has  collected  at  one  point,  into  sixteen  to 
twenty-four  spores.  In  addition  to  the  asexual  forms, 
sexual  forms  are  also  seen,  which  resemble  those  of  the 
tertian  parasite. 

The  Tropical  Parasite  (Plate  XIV,  Fig.  X).— During 
the  rise  of  the  fever,  especially  in  the  first  attack,  no  para-, 
sites  are  found,  or  at  most  only  occasional  blue  rings, 
which  are  much  smaller  than  the  small  tertian  rings,  and 
have  no  crescentic  thickening  at  their  periphery  (small 
tropical  rings).  Their  further  development,  in  contrast 
to  the  other  forms  of  malaria,  does  not  take  place  in  the 
circulating  blood,  but  in  the  inner  organs  (spleen,  brain, 
bone-marrow),  so  that  division  forms  are  not  seen  in 
specimens  made  from  the  blood.  The  appearance  of  the 
gametes,  which  are  crescentic,  is  typical  of  the  tropical 
parasite.  They  stain  more  intensely  at  the  poles  than  in 
the  centre,  at  which  the  pigment  is  arranged  in  the  form 
of  a  wreath. 

The  Differential  Diagnosis  between  the  different  forms  of 
malaria  can  be  made  with  certainty  only  when  careful 
measurement  of  the  temperature  accompanies  the  micro- 
scopical examination.  In  doubtful  cases  the  temperature 
must  be  taken  every  three  hours,  day  and  night.  The  ter- 
tian parasite  is  characterized  by  the  large  pigmented  rings 
and  by  the  enlargement  of  the  erythrocytes ;  the  quartan 
by  the  band  forms  and  the  absence  of  enlargement  of  the 
erythrocytes;  the  tropical  by  the  crescents.  If  the  para- 
sites are  found  in  the  microscopical  specimen  only  in  the 
form  of  small  rings,  it  is  impossible  to  make  a  diagnosis 
from  the  microscopical  examination  alone,  since  the  small 
tertian  and  quartan  rings  have  the  same  appearance,  and 
resemble  the  large  tropical  rings.  Further,  the  possibility 


268    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

of  mixed  infection  must  be  remembered,  as  well  as  the 
alteration  in  the  appearance  of  the  different  forms,  due  to 
the  ingestion  of  quinine  before  the  paroxysm  (quinine 
forms) . 

(J)  Spirilla  of  Relapsing  Fever  (Plate  XV,  Fig.  Y).- 
For  the  detection  of  spirilla  of  relapsing  fever  the  blood 
must  be  obtained  during  the  fever,  since,  as  a  rule,  the 
spirilla  appear  in  the  circulating  blood  but  a  few  hours 
before  the  rise  of  the  fever. 

The  blood  is  collected  and  the  smears  prepared  in  the 
same  manner  as  for  the  detection  of  malarial  parasites. 

The  staining  is  done  with  the  diluted  Giemsa  stain 
(one  drop  to  one  cc  of  distilled  water) .  The  examination 
of  the  fresh  specimen  is  best  done  with  dark  illumination. 

The  spirilla,  discovered  by  Obermeyer,  are  highly 
motile,  very  fine  spiral  threads,  with  pointed  ends,  10  to 
40  /*  in  length  and  1  /*  in  thickness.  They  usually  lie 
singly  or  a  few  side  by  side,  and  rarely  form  snarls. 

In  order  to  examine  the  blood  for  trypanasomes  the 
specimen  is  stained  in  the  same  way  as  for  the  examina- 
tion for  malaria. 

2.  The  Examination  of  the  Blood  by  Means  of  Culture  Media 

The  most  important  bacteria,  whose  presence  in  the 
blood  is  demonstrated  by  means  of  culture  media  are: 
strepto-,  staphylo-,  pneumo-,  and  gonococci;  typhus-, 
paratyphus-,  coli-,  pyocyaneus-,  plague-  and  anthrax 
bacilli.  For  the  purpose  of-  making  cultures  it  is  best  to 
take  the  blood  from  the  median  vein  by  puncture. 

The  arm  hanging  down  at  the  side  of  the  body  is  tied 
with  a  bandage  or  something  similar  above  the  elbow,  the 
elbow  region  over  the  median  vein  is  first  thoroughly 
washed  with  soap  then  with  alcohol,  ether,  and  sublimate 
and  the  sublimate  is  removed  by  washing  with  freshly 


BLOOD  269 

boiled  water.  The  freshly  boiled  cannula  of  the  syringe  is 
plunged  into  the  vein,  which  stands  out  prominently,  and 
the  blood  is  aspirated.  The  bandage  is  removed  from  the 
arm  before  the  syringe  is  withdrawn. 

The  blood  may  also  be  removed  by  means  of  a  sterile 
cup  or  by  puncturing  the  finger  or  the  ear.  The  latter 
method  has  the  drawback,  that,  as  a  rule,  the  blood 
becomes  contaminated  by  skin  cocci  and  that  we  cannot 
always  get  a  sufficient  quantity.  The  blood  is  permitted 
to  drop  directly  on  the  culture  medium  or  we  can  carry  it 
to  the  culture  medium  with  a  sterile  pipette. 

Usually  3  to  5  cc  of  blood  are  obtained,  which  is  at 
once  put  into  a  small  flask  with  50  to  100  cc  of  bouillon, 
or  is  mixed  with  melted  agar  which  has  cooled  down  to 
45°  and  which  is  immediately  poured  over  plates.  Can- 
non recommends  to  squirt  the  blood  into  glasses  of  slant- 
ing agar  upon  which  it  coagulates  after  it  has  been  equably 
distributed  by  proper  motions  and  after  the  glass  has  been 
put  in  a  slanting  position.  The  cultures  are  put  into  the 
incubator  for  twenty- four  hours  and  then  examined.  Then 
smears  are  made  on  agar  plates  from  the  bouillon. 

We  have  to  be  especially  careful  when  we  find  staphy- 
lococci,  as  even  after  the  most  careful  operation  the  cocci 
may  have  come  from  the  skin.  Such  possibility  is  espe- 
cially to  be  considered,  when  the  cultures  of  the  blood  show 
staphylococci,  while  different  germs  are  found  in  the  pus. 
In  order  to  determine  whether  we  are  dealing  with  patho- 
genic staphylococci  we  can  make  the  agglutination  test 
and  examine  for  toxine  formation. 

Blood  cultures  of  the  typhoid  and  paratyphoid  bacilli 
are  made  in  the  media  of  Conradi  and  Kayser  to  which 
bile  has  been  added.  Conradi' s  medium  consists  of  fresh 
bovine  bile,  to  which  is  added  10  per  cent,  peptone  and 
10  per  cent,  glycerine;  of  this  5  cc  are  put  into  test-tubes 


270     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

and  the  test-tubes  are  sterilized  in  live  steam  for  two  hours. 
Kayser  uses  merely  the  gall  alone,  without  any  further 
addition.  These  sterilized  tubes  must  be  kept  cool.  About 
2.5  cc  of  blood  are  put  into  such  a  test-tube.  However, 
even  smaller  quantities  of  blood  may  give  positive  results. 
These  gall  media  enable  us  also  to  make  cultures  from  the 
coagulated  blood  for  the  typhoid  and  paratyphoid  bacilli, 
and  we  can  use  for  such  purpose  the  blood-cakes  of  blood 
specimens  which  have  been  used  for  making  the  Gruber- 
Vidal  test.  The  blood-cake  is  transferred  directly  into 
the  gall  medium  of  the  test-tube  after  pouring  off  the 
serum.  By  such  cultures  we  are  able  to  make  an  early 
diagnosis  at  a  time  when  the  agglutination  test  is  negative. 

The  test-tubes  with  gall  media  to  which  the  blood  has 
been  added,  are  put  into  the  incubator  for  fourteen  to 
twenty  hours.  Without  shaking  the  test-tubes  a  few  loops 
are  carefully  removed  from  the  surface  of  the  gall  media, 
and  after  this  a  larger  quantity,  about  0.5  cc,  which  are 
transplanted  upon  a  large  Endo-  or  Conradi-Drigalslci 
plate,  and  rubbed  into  this  with  a  glass  spatula.  The 
examination  of  these  media  is  made  in  the  usual  manner. 

The  typhoid  bacilli  are  found  in  the  blood  during  the 
entire  febrile  stage,  very  often  already  in  the  first  days  of 
the  disease  and  are  especially  numerous  during  the  stage 
of  the  eruption  of  the  roseolse.  During  the  afebrile  stage 
they  cannot  be  demonstrated.  Likewise,  after  the  febrile 
state  is  gone,  an  attempt  to  prove  their  presence  is  often 
fruitless.  Even  at  the  height  of  the  fever  we  may  not  be 
successful  if  the  disease  is  of  a  mild  type. 

A  culture  may  be  made  not  only  from  the  circulating 
blood,  but  also  from  the  roseolse  in  which  they  are  always 
found.  The  cultures  are  made  in  the  following  way  after 
Neufelds:  The  skin  is  first  washed  with  a  mixture  of  even 
parts  of  alcohol  and  ether,  a  very  slight  incision  is  made 


BLOOD  271 

with  a  sharp  scalpel  into  the  roseola,  a  little  matter  is 
scraped  out  with  the  scalpel  and  put  at  once  into  bouillon. 
The  incision  must  be  made  so  superficially  that  no  blood 
should  come,  as  the  typhoid  bacilli  are  in  the  tissue  of  the 
roseola  and  not  in  the  blood.  Should  blood  come  then  a 
little  bouillon  is  dropped  on  the  wound  so  as  to  dilute  the 
blood  immediately.  This  bouillon  diluted  blood  is  im- 
mediately transferred  into  the  bouillon  test-tubes.  The 
test-tubes  are  the  nput  into  the  incubator  which  is  kept 
at  37°  C. ,  for  eight  hours,  after  which  time  smears  are  made 
on  agar,  and  the  bacterial  growth  is  examined  the  next  day 
by  means  of  agglutination,  vaccination  of  litmus-whey, 
etc.  In  the  bouillon  test-tubes  are  found  usually  staphy- 
lococci  besides  the  typhoid  bacilli. 

The  newly  appearing  roseolas  are  best  suited  for  ex- 
amination, and  by  preference  several  of  them  ought  to  be 
examined  at  the  same  time.  Several  specimens  are  taken 
from  each  roseola  and  implanted  into  the  bouillon  test- 
tubes.  Good  results  were  obtained  also  by  ' Schmiedecke 
who  followed  the  method  of  Neufelds  with  the  variation 
that  he  removed  the  skin  over  the  roseolas  in  very  fine 
layers  and  planted  these  in  the  bouillon. 

Instead  of  using  bouillon  the  test-tubes  containing  the 
gall  media  can  also  be  used. 

(3)  EXAMINATION  OF  THE  BLOOD  BY  MEANS  OF  ANIMAL 

INOCULATION 

Animal  inoculation  is  of  special  value  for  the  detection 
of  anthrax  and  plague  bacilli  in  the  circulating  blood. 

Anthrax  Bacilli. — White  mice  or  guinea-pigs  are  used 
as  test-animals,  and  are  inoculated  subcutaneously  with 
!  to  0.3  or  even  1.0  cc  of  the  blood  obtained  by  vene- 
puncture.     If  the  blood  contains  anthrax  bacilli,  the  ani- 
mals die  of  anthrax  septicaemia,  and  the  bacilli  can  be 


272     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

detected  in  the  blood  and  viscera  by  microscopical  and 
cultural  examination. 

Plague  Bacilli— Rats  or  guinea-pigs  are  inoculated  with 
blood. 

Animal  inoculation  may  also  be  used  for  the  detection 
of  STREPTOCOCCI  in  the  blood.  It  is  not,  however,  as 
reliable  as  are  cultural  procedures,  since  streptococci 
which  are  highly  virulent  for  man  may  be  avirulent  for 
animals,  so  that  a  negative  result  does  not  necessarily  ex- 
clude the  possibility  of  the  presence  of  streptococci  in  the 
blood.  White  mice  are  used  as  test-animals,  and  are  in- 
oculated intraperitoneally  with  0.5  to  1.5  cc  of  blood. 

If  the  blood  contains  streptococci  virulent  for  mice, 
the  animals  die  of  streptococci  septicaemia. 

(4)  SERUM  DIAGNOSIS. 

Serum  diagnosis  depends  upon  the  fact  that  specific 
reaction  products— agglutinins  and  bacterioly sins— appear 
in  the  blood  of  persons  who  are  suffering  or  have  suffered 
from  infectious  diseases.  This  observation  has  found 
practical  application  principally  in  \the  diagnosis  of 
typhoid  fever.  Shortly  after  Grueber  h^d  determined  that 
the  blood  of  patients  convalescing  from  typhoid  fever  has 
the  power  to  agglutinate  typhoid  bacilli,  Widal  called 
attention  to  the  fact  that  the  blood-serum  contains  the 
agglutinins  even  during  the  course  of  the  disease,  and 
often  in  its  first  stage. 

Performance  of  the  Agglutination  Test  (the  Widal 
Reaction) 

The  blood  is  best  obtained  by  venepuncture  or  cupping 
—about  2  cc  are  taken.  If  these  methods  are  impracti- 
cable, the  blood  is  obtained  from  a  prick  in  the  ball  of  the 


BLOOD  273 

finger,  and  collected  in  a  small  centrifuge  tube.     After 
the  blood  has  coagulated,  the  clot  is  loosened  with  a  sterile 
platinum  needle  from  the  sides  of  the  tube.     In  the  course 
of  the  next  few  hours,  during  which  the  blood  is  kept  in 
an  ice-chest,  sufficient  serum  is  usually  obtained.     This 
is  removed  with  a  pipette,  diluted  ten  times  with  a  sterile 
08.5  per  cent,  solution  of  sodium  chloride  (1  part  serum 
and  9  parts  salt  solution) ,  and  centrifugalized  until  clear. 
From  this  further  dilutions,  in  the  ratios  of  1:  20,  1:40, 
1 :  50,   1 :  60,  etc. ,  are  made.       In  1  cc  of  each  dilution 
one  loop  of  an  eighteen  to  twenty-four  hours'  agar  typhoid 
culture,   whose   agglutination-titre    (reaction),   with    an 
artificial  typhoid  immune  serum,  is  known,  is  carefully 
mixed,  according  to  the  method  described  on  p.  116.      If 
agglutination  does  not  take  place  at  once,  the  inoculated 
tubes  are  kept  one  hour  at  37°  C.  and  again  examined. 
They  are  examined  macroscopically  for  clumping,  in  the 
manner  described  on  p.  116. 

It  is  always  necessary  to  make,  simultaneously,  con- 
trols with  the  salt  solution  used  as  diluent  and  with  nor- 
mal human  serum.  The  solution  in  the  control-tubes 
must  remain  evenly  turbid  during  the  period  of  observa- 
tion. It  is  always  necessary  to  titrate  the  serum— that 
is,  to  determine  in  how  great  a  dilution  it  still  causes 
agglutination.  In  regard  to  the  value  of  the  results  of 
this  test,  it  must  be  remembered  that  the  serum  of  healthy 
persons  who  have  never  had  typhoid  fever  may  cause  the 
agglutination  of  typhoid  bacilli.  Experience  has,  how- 
ever, shown  that  this  is  true  only  when  the  serum  is  highly 
concentrated.  When  the  test  is  carried  out  in  the  above- 
described  manner,  normal  serum  does  not  cause  aggluti- 
nation in  dilutions  over  1  :  50.  Therefore,  if  agglutina- 
tion can  be  seen  macroscopically  in  the  inoculated  tubes 
containing  dilutions  over  50  within,  at  the  longest,  one 


274    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

hour's  stay  in  an  incubator  at  87°  C.,  while  the  controls 
(with  sodium  chloride  and  normal  serum  in  a  dilution  of 
1 : 50)  appear  homogeneous,  it  can  be  assumed  that  in  all 
probability  the  patient  has  typhoid  fever,  or  has  recently 
had  it. 

In  reporting  the  result  of  the  agglutination  test  it  is 
not  sufficient  to  speak  of  a  positive  or  negative  reaction, 
but  rather  the  limit  of  the  agglutinating  power  of  the 
serum,  its  titre  (potency),  and  the  method  by  which  the 
latter  is  estimated,  should  be  given.  The  latter  is  neces- 
sary, because  a  number  of  investigators  establish  the 
appearance  of  agglutination  microscopically  with  the  high 
power,  and  not  macroscopically,  and  consider  the  clump- 
ing of  a  few  bacilli  as  evidence  of  agglutination.  These 
authors  must  naturally,  in  order  to  avoid  mistakes,  set 
the  lowest  limit  of  the  agglutination  much  higher  (1:100) 
than  is  necessary  with  the  macroscopical  examination. 

The  diagnostic  value  of  the  Widal  reaction  is  still 
further  handicapped  by  the  fact  that  it  rarely  appears  at 
the  beginning  of  the  disease,  and  in  a  number  of  cases  is 
absent  during  its  entire  course.  As  a  rule  the  agglutinins 
cannot  be  detected  until  during  the  second  week  of  the 
disease.  From  this  it  follows  that  a  negative  reaction 
never  excludes  the  possibility  of  typhoid  fever.  The 
agglutination  test  cannot  replace  the  direct  detection  of 
the  bacilli.  The  appearance  of  agglutinins  in  the  blood 
is  to  be  considered  merely  as  a  symptom  of  typhoid  fever. 
Their  presence  strengthens  the  diagnosis,,  but  their  absence 
does  not  shake  it. 

Picker  has  recently  endeavored  to  simplify  the  execu- 
tion of  the  agglutination  test  in  order  to  place  it  in  the 
hands  of  the  practitioner  who  has  no  laboratory  equipment 
at  his  disposal.  His  "typhoid-diagnosticum,"  which 
replaces  the  living  typhoid  culture,  consists  of  a  mixture 


BLOOD  275 

of  dead  typhoid  bacilli.  It  is  a  slightly  turbid,  sterile 
fluid,  which  keeps  a  long  time  fit  for  use  if  kept  cool  in 
the  dark,  and  if  shaken  from  time  to  time.  It  must 
always  be  shaken  before  using. 

" The  test  is  carried  out  in  the  following  manner:  A 
dilution  of  1 :10  of  the  serum  to  be  tested  with  sterile  0.85 
per  cent,  sodium  chloride  solution,  is  made  by  means  of 
a  graduated  pipette,  and,  for  example,  0. 2  and  0. 1  cc  of 
this  dilution  are  placed  in  conical  test-tubes.  To  tube  1, 
0.85  cc  of  the  'diagnosticum'  is  added;  to  tube  2,  0.9  cc. 
A  third  tube  receives  1  cc  of  the  'diagnosticum'  without 
the  addition  of  serum  (control).  The  tubes  are  closed 
with  a  cork  or  rubber  stopper,  the  contents  thoroughly 
mixed,  and  set  aside  at  room-temperature  and  protected 
from  the  light.  The  result  is  evident  after  ten,  twelve, 
or  fourteen  hours.  The  determination  of  the  result  must 
not  be  postponed  more  than  twenty  hours.  The  observa- 
tion of  the  contents  of  the  tubes  is  simplified  if  they  are 
examined  against  a  black  background,  or  if  the  outspread 
hand  is  held  5  to  10  centimetres  behind  the  tube,  which  is 
raised  to  the  level  of  the  eye,  and  between  it  and  the  source 
of  light  (window) .  Positive  agglutination  is  made  evi- 
dent by  clarification,  and  simultaneous  clumping  of  the 
agglutinins  contained  in  the  specimen,  which  is  particu- 
larly well  seen  owing  to  the  use  of  a  conical  test-tube. " 

The  control,  which  contains  the ' '  diagnosticum' '  alone, 
must,  of  course,  remain  evenly  turbid. 

The  agglutination  test  is  not  applicable  for  the  early 
diagnosis  of  other  infectious  diseases,  as  cholera  and 
plague. 

The  examination  of  the  blood  for  bacteriolysins  has 
been  used  in  the  so-called  Pfeiffer's  test  (cf.  p.  122)  for 
the  recognition  of  convalescing  cases  of  typhoid  and 
cholera.  The  detection  of  the  specific  bacteriolysins  has 


276     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

not  been  used  in  the  diagnosis  of  new  cases  up  to  the  pres- 
ent time.  Stern l  has  recently  used  it  in  the  diagnosis  of 
typhoid  fever.  His  results,  however,  have  not  as  yet  been 
tested  with  a  large  amount  of  material.  He  tested  the 
serum  of  patients  suspected  of  having  typhoid  fever\  with 
the  aid  of  the  test-tube  reactions  of  bactericidal  substances, 
according  to  the  methods  worked  out  by  Elirlicli  and  his 
scholars. 

It  is  determined  what  the  smallest  dose  is  in  which 
the  serum  to  be  examined  still  has  bactericidal  action. 
For  this  purpose  a  constant  quantity  of  typhoid  bacilli 
and  of  normal  complementary  serum  is  added  to  de- 
creasing quantities  of  serum  which  has  been  rendered 
inactive. 

Stern  used  0.5  cc  of  a  fresh  1:  10-15  dilution  of  nor- 
mal rabbit  serum  as  complement;  for  inoculation,  0.5  cc 
of  a  dilution  of  1:  5,000  of  a  twenty-four-hour  typhoid 
bouillon  culture.  The  sera  are  diluted  with  0. 85  per  cent, 
sodium  chloride  solution;  the  culture  is  diluted  with 
bouillon. 

The  test  is  performed  in  the  following  manner:  The 
serum  to  be  examined  is  rendered  inactive  by  heating  for 
half  an  hour  on  a  water-bath  at  55°  C.  Quantities  of  1.0, 
0.8,  0.1,  0.03,  0.01  cc,  etc.,  are  placed  in  a  series  of 
tubes  by  means  of  a  sterile  1  cc  graduated  pipette.  If  it 
is  suspected  that  the  serum  is  of  high  potency,  dilutions 
of  1 : 50-100  are  made  at  once.  In  addition,  each  tube 
receives  0.5  cc  of  a  1:12  dilution  of  fresh  rabbit  serum, 
and  0.5  cc  of  a  1:  5,000  typhoid  bouillon  culture.  The 
tubes  are  then  filled  to  the  same  level — 2  cc — with  0.85 
per  cent,  sodium  chloride  solution.  It  is  necessary  to 
make  the  following  controls : 

1  Berliner  Klin.  Wochenschr.,  1904,  No.  3. 


BLOOD  277 

Two  controls  of  the  typhoid  culture  (I  and  II),  each 
of  which  contains  1.5  NaCl  +  0.5  cc  of  a  1 :  5,000  typhoid 
bouillon  culture. 

A  plate  is  made  at  once  from  Control  I,  another  after 
three  hours  at  37°  C.  from  Control  II. 

Controls  III  and  IV  establish  the  inactivity  of  the 
maximal  dose  used  of  the  immune  serum  which  has 
been  rendered  inactive,  and  of  the  complementary  serum 
alone. 

Control  III:  1.0  of  immune  serum  which  has  been 
rendered  inactive. +0.5  cc  of  1:5,000  typhoid  culture 
+0.5  cc  NaCl. 

Control  IV:  0.5  rabbit  serum  +0.5  cc  of  1:5000 
typhoid  culture  +1.0  cc  NaCl. 

Controls  V  and  VI  test  the  sterility  of  the  maximal 
quantity  of  the  two  sera  used. 

Control  V:  1.0  cc  of  the  inactive  immune  +1  0  cc 
NaCl. 

Control  VI:  0.5  cc  of  rabbit  serum  +1.5  cc  NaCl. 

All  the  tubes,  with  the  exception  of  Control  I,  are 
after  being  shaken,  placed  for  three  hours  in  an  incubator 
at  37°  C.     After  this  length  of  time  they  are  again  shaken, 
and  agar  plates  made  from  them  by  mixing  their  contents 
with  melted  agar  which  has  been  cooled  to  42°  C     and 
pouring  the  latter  into  Petri  dishes.     The  plates  are  placed 
inverted,  in  an  incubator,  and  remain  there  until  the  fol- 
lowing day,  when  it  is  determined  how  many  colonies  have 
developed  in  the  individual  plates.     The  estimation  is 
made  according  to  the  following  scheme:  0,  or  almost  0 
about  one  hundred,   a  ^few  hundreds,  thousands,  many 
thousands,  innumerable  colonies.     In  the  examination  of 
the  plates  it  is  noticed  that  those  containing  the  largest 
quantities  of  immune  serum  show  the  most  colonies.     This 


278    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

fact  is  due  to  the  diversion  of  the  complements  by  super- 
fluous immune  bodies. 

A  distinct  bactericidal  action  is  present  only  when  the 
controls  tally,  and  there  is  reduction  of  the  colonies  from 
innumerable,  or  many  thousands,  to  0,  or  very  few. 

Still  further,  the  test  is  to  be  considered  as  positive 
only  when  the  lowest  limit  of  the  active  serum  dilution 
has  been  reached — that  is,  when  the  last  plates  show  an 
increasing  number  of  colonies  (Neisser) . 

The  Serum  Diagnosis  of  Syphilis  According  to 
Wassermann 

The  positive  result  of  the  Wassermann  reaction  indi- 
cates that  the  individual  furnishing  the  examined  serum 
is  or  has  been  infected  with  syphilis.  The  theory  of  the 
reaction  is  based  on  the  observation  that  a  combination 
of  antigen  and  antibody  will  bind  complement,  whereas 
neither  antigen  nor  antibody  alone  possesses  any  or  a  very 
slight  affinity  for  complement. 

A  hemolytic  system  (red  blood-cells  +  serum,  obtained 
from  a  rabbit  previously  injected  with  red  blood-corpuscles 
serves  as  an  indicator  for  the  complement  fixation.  If 
we  add  a  hemolytic  system  to  a  solution  containing  anti- 
gen +  antibody  and  complement,  hemolysis  does  not  take 
place,  because  of  the  complement  entering  into  the  antigen 
+  antibody  complex,  whereas  hemolytic  amboceptor  and 
complement  are  required  for  the  production  of  hemolysis. 
If,  on  the  other  hand,  the  solution  contains  either  antigen 
or  antibody  and  complement,  the  complement  cannot  be 
fixed,  and  it  remains  free  to  produce  solution  of  the  red 
blood-corpuscles  of  the  added  hemolytic  system.  The 
absence  of  hemolysis,  therefore,  shows- a  positive  reaction, 
and  its  presence  indicates  a  negative  reaction. 


BLOOD  279 

In  the  examination  for  antibodies,  which  are  traceable 
to  luetic  infection,  blood  serum  is  used;  in  parasyphilitic 
diseases  of  the  central  nervous  system  lumbar  fluid  is  used. 
A  specially  prepared  extract  of  the  livers  of  congenitally 
syphilitic  infants  is  used  as  antigen,  the  livers  having  been 
previously  tested  as  to  their  fitness  for  this  purpose.  The 
method  of  procedure  is  as- follows: 

The  liver  extract  is  added  to  the  previously  inactivated 
serum,  complement  consisting  of  guinea-pig  serum  is 
then  added,  and  the  mixture  is  put  into  the  incubator  for 
one  hour,  to  permit  the  action  between  antibody,  antigen, 
and  complement  to  take  place.  On  the  expiration  of  this 
time,  the  hemolytic  system  is  added,  and  the  tubes  are 
again  allowed  some  time  in  the  thermostat,  and  thereupon 
are  set  in  the  refrigerator  overnight.  The  result  of  the 
test  may  then  be  determined.  If  the  examined  serum 
contains  antibodies,  that  is,  if  the  result  be  POSITIVE, 
hemolysis  lias  not  taken  place,  the  red  blood-corpuscles  are 
collected  at  the  bottom  of  the  tube  and  the  supernatant 
liquid  is  colorless;  in  the  absence  of  antibodies,  that  is, 
if  the  result  is  NEGATIVE,  this  is  shown  by  the  fact 
that  solution  lias  taken  place,  the  tube  contains  no  sedi- 
ment of  red  blood-corpuscles,  but  the  entire  almost  clear 
liquid  has  become  red,  colored  by  the  dissolved  blood 
pigment. 

Method  of  Applying  the  Reaction. — The  blood  necessary 
for  the  reaction  is  obtained  by  a  venous  puncture,  the 
median  basilic  vein  being  the  best  for  the  purpose,  or  by 
the  use  of  a  cupping  cup.  Six  to  eight  cc  are  taken  from 
adults,  and  1-J-  to  2  cc  from  children.  The  blood  is 
received  into  sterile  centrifuge  tubes,  and  after  clotting, 
it  is  separated  from  the  walls  of  the  tube  by  means  of  a 
sterile  needle  and  centrifugalized  to  obtain  the  required 
serum.  The  separated  serum  is  decanted  and  immedi- 


280    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

ately  inactivated  (complement  destroyed  by  heating  it 
on  a  water  bath  to  56°  C.  for  half  an  hour). 

Two  control  sera,  similarly  inactivated,  are  always  ex- 
amined simultaneously  with  the  suspected  serum,  one 
from  a  known  syphilitic  serum,  rich  in  antibodies,  and 
the  other  a  known  normal  serum.  The  serum  must  be 
examined  in  as  fresh  a  state  as  possible,  as  syphilitic 
serum  which  has  been  kept  for  a  time  may  lose  its  prop- 
erty of  binding  complement,  and  vice  versa;  normal 
serum  may  develop  this  property.  Two  specimens  of  the 
suspected  serum,  namely,  0.2  cc  and  0.1  cc,  and  one  each 
of  0.2  cc  of  the  control  sera,  are  employed. 

Antigen. — If  the  liver  furnishing  the  antigen  is  not 
to  be  used  at  once,  it  may  be  kept  in  a  Morgenroth  refrig- 
erator in  a  frozen  condition. 

Preparation  of  the  Aqueous  Extract  ( Wassermann  and 
G.  Meyer) .  The  liver  is  cut  into  small  particles,  with  a 
knife  and  scissors,  mixed  with  four  parts  of  physiologic 
salt  solution,  to  which  phenol  in  the  proportion  of  0.5 
per  cent,  is  added,  and  the  mixture  agitated  in  the 
shaking  apparatus  for  twenty-four  hours.  For  example, 
360  cc  salt  solution  (0.85  per  cent.),  100  gms.  liver, 
40  cc  phenol,  0.5  per  cent.  The  resulting  mass  is  centri- 
fugalized  until  a  perfectly  clear  extract  is  obtained. 

Alcoholic  Extract. — Michaelis  furnishes  the  following 
formula  for  its  preparation:  "The  liver  is  minced  in  a 
mortar,  ten  volumes  of  absolute  alcohol  are  immediately 
added,  and  the  mixture  shaken  with  the  aid  of  glass  beads 
for  ten  to  twelve  hours.  At  the  expiration  of  twenty-four 
hours,  the  clear  liquid  is  removed  from  the  sediment  with 
a  pipette,  and  preserved  in  the  refrigerator,  to  be  used  as 
stock  solution  (antigen) .  For  each  test  of  the  reaction, 
a  freshly  prepared  dilution  of  the  stock  solution  with 
four  parts  of  physiologic  salt  solution  is  employed.  This 


BLOOD  281 

alcoholic  stock  solution  forms  a  slightly  milky  emulsion 
with  the  aqueous  solution.  Its  slight  alcoholic  content 
does  not  interfere  with  the  reaction.  On  allowing  this 
milky  emulsion  to  stand,  a  flocculent  precipitate  gradually 
settles  on  the  bottom;  the  clear  supernatant  liquid  is  then 
ineffective,  as  the  active  principle  is  fixed  in  the  precipitate 
and  the  emulsion  must  be  well  shaken  to  render  it  active 
again.  One  cc  of  this  diluted  emulsion  is,  as  a  rule,  em- 
ployed for  the  reaction,  although  considerable  latitude  as 
to  the  quantity  of  liver  extract  is  permitted." 

Alcoholic  extracts  of  normal  organs,  prepared  in  the 
above-described  manner,  have  been  recommended  as  prac- 
ticable antigen.  Fleischmann  found  four  out  of  five  ex- 
tracts of  normal  livers  available.  Michaelis  has  used  a 
very  effective  alcoholic  extract  of  normal  (human)  hearts. 
It  is  better,  however,  at  the  present  stage,  at  least,  to  use 
the  extract  of  syphilitic  livers  in  order  to  obtain  unobjec- 
tionable results. 

Previous  to  its  employment,  each  extract  must  be  test- 
ed as  to  its  availability  by  comparison  with  a  known 
syphilitic  and  a  known  normal  serum,  as  available  ex- 
tracts are  not  always  obtained,  even  from  syphilitic  fetal 
livers. 

Antigen  is  considered  particularly  efficacious  if  0. 1  cc  + 
0. 1  cc  of  syphilitic  serum  inhibits  hemolysis  completely. 
Antigens,  of  which  0.2  to  0.4  cc  +  0.1  to  0.2  cc  of  luetic 
serum  are  required  for  complete  inhibition,  may  also  be 
used,  provided  they  do  not  produce  inhibition  either 
with  normal  sera  or  by  themselves  in  double  amount  (G. 
Meyer) . 

For  the  preliminary  test  of  the  extract,  following  the 
suggestion  of  Meyer ,  the  smallest  amount  of  extract,  which 
by  itself  will  bind  complement,  is  determined  by  testing 
with  0.2,  0.4,  0.6,  0.8  and  1.0  cc  of  antigen;  and  further- 


282     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

more,  the  smallest  dose  which  will  cause  complete  inhi- 
bition with  0. 1  cc  of  luetic  serum  is  noted. 

The  extracts,  when  kept  cool  and  dark,  often  remain  ser- 
viceable for  several  months,  but  they  may  suddenly  become 
ineffective;  for  this  reason  it  is  imperative  that  their 
efficacy  be  controlled  at  each  test  with  a  known  luetic  and 
a  known  normal  serum.  In  making  the  test,  two  corre- 
spondingly different  doses  of  the  extract  and  the  serum 
are  used,  as,  for  instance,  0.2  cc  of  antigen  +  0.2  cc  of 
serum,  and  0.1  cc  of  antigen  +0. 1  cc  of  serum;  the 
amounts  used  depend  upon  the  result  of  the  preliminary 
examination. 

Complement. — Fresh  guinea-pig  serum  serves  as  comple- 
ment. One  cc  of  a  10-per-cent.  dilution  in  physiologic 
salt  solution  is  required.  The  serum  may  be  conserved 
for  a  short  time  (not  more  than  forty-eight  hours)  in  the 
ice-chamber.  Guinea-pig  blood  may  be  obtained  by  heart 
puncture. 

Hemolytic  System. — Red  blood-corpuscles  (erythrocytes) 
of  the  sheep,  and  serum  obtained  from  a  rabbit  previously 
treated  by  repeated  injections  of  washed  sheep's  blood- 
corpuscles,  constitute  thehemolytic  system.  The  sheep's 
blood  is  obtained  by  puncture  of  the  jugular  vein  and  re- 
ceived in  salt  solution.  The  blood-corpuscles  are  sepa- 
rated by  centrifugalizing,  and  washed  several  times  with 
physiologic  salt  solution.  The  blood-corpuscles  are  shaken 
with  the  salt  solution  in  the  centrifuge  tube,  then  centri- 
fugalized  and  the  supernatant  liquid  is  poured  off;  this  is 
repeated  several  times.  For  the  test,  1  cc  of  a  5-per- 
cent, suspension  of  blood-corpuscles  in  physiologic  salt 
solution  is  used. 

The  hemolytic  serum  keeps  well  for  some  time,  if  put 
into  a  dark  bottle  and  placed  in  the  refrigerator.  For 
the  test,  1  cc  of  a  dilution,  which  corresponds  in  solving 


BLOOD  283 

power  to  a  serum  capable  of  dissolving  a  two  and  a  half 
to  threefold  dose  of  the  serum,  is  employed. 

Its  titre  must  be  determined  before  instituting  the  re- 
action. For  trial-tests,  G.  Meyer  recommends  one  and 
one-half,  two-  and  threefold  strengths  of  that  dilution 
with  which  good  results  were  obtained  at  the  last  test.  In 
order  to  adapt  the  result  of  the  trial-test  to  the  condi- 
tions of  the  actual  test,  the  complement,  with  the  addi- 
tion of  the  blood-corpuscle  serum  mixture  and  2  cc  of 
physiologic  salt  solution  is  placed  in  the  incubator  for 
one  hour. 

If  the  titre  of  the  serum  has  remained  unchanged,  com- 
plete hemolysis  will  be  found  to  have  taken  place  in  the 
tube  containing  the  one  and  one-half  dilution  within  half 
an  hour,  and  in  the  tube  containing  the  twofold  dilution 
at  the  latest  within  one  hour.  The  threefold  dilution  is, 
as  a  rule,  completely  hemolyzed  within  two  hours,  but  that 
result  need  not  be  awaited  in  order  to  proceed  with  the 
reaction. 

For  the  Determination  of  the  Reaction  the  Following:  Are 
Necessary: 

1.  Antigen  in  two  doses,  i.e.,  0.1  and  0.2. 

2.  The  serum  to  be  examined,  0.5  cc. 

3.  Known  luetic  serum,  0.4  cc. 

4.  Known  normal  serum,  0.4  cc. 

5.  Fresh  guinea-pig  serum  (1:10  NaCl  solution). 

6.  Five  per  cent,  suspension  of  sheep's  erythrocytes  in 
physiologic  salt  solution. 

7.  Rabbit  serum,  hemolytic  for  sheep's  erythrocytes. 

8.  Physiologic  salt  solution. 

The    test-tubes    should  have  a  capacity  of  at  least  5 
cc,  and  are  eventually  rilled  up  to  that  amount  with  physi- 


284     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

ologic  salt  solution.  For  the  purpose  of  avoiding  with 
certainty  any  possible  source  of  error,  a  row  of  control  test- 
tubes  are  employed  in  addition  to  the  tubes  containing 
the  suspected  sera  (tubes  I  and  II) . 

1.  A  control  with  a  double  dose  of  antigen,  to  prove 
that  the  antigen  itself  produces  no  fixation  (tube  III) . 

2.  The  positive  control:  Antigen  and  known  luetic 
serum  (tube  IV) . 

8.  The  negative  control:  Antigen  and  known  normal 
serum  (tube  V) . 

The  controls  in  tubes  III,  IV,  and  V  serve  to  demon- 
strate the  fitness  of  the  antigen. 

4.  Controls  to  show  that  the  sera  themselves  do  not  fix 
complement  (tubes  VI,  VII,  and  VIII) . 

5.  A  tube  containing  NaCl  solution,  complement  and 
blood-corpuscles  is  employed  to  prove  that  the  complement 
itself  possesses  no  hemolytic  properties  (tube  IX) . 

6.  Control  of  the  hemolytic  system  (tube  X) . 

7.  A  tube  containing    salt  solution    and  red  blood- 
corpuscles  in  NaCl  solution  (tube  XI) . 

In  making  the  test,  the  tubes  containing  serum,  anti- 
gen, and  complement  are  placed  in  the  incubator  for  one 
hour  to  produce  fixation  of  the  complement.  Thereupon 
the  sheep  erythrocytes  and  the  hemolytic  serum,  both  of 
which  have  previously  been  kept  for  about  one-quarter  to 
one-half  an  hour  at  a  temperature  of  37°  C. ,  are  added  to 
each  tube. 

The  course  of  the  reaction  must  now  be  watched  care- 
fully; as  soon  as  hemolysis  is  complete  in  the  control-tubes, 
the  test-tubes  are  taken  from  the  thermostat  and  placed 
on  ice  for  twenty  hours. 

The  determination  of  the  reaction  with  an  antigen, 
the  efficient  dose  of  which  has  been  determined  as  0. 1 


BLOOD 


285 


cc,  and  giving  a  positive  result,  is  illustrated  by  the  fol- 
lowing table : 


Serum 

««  • 

o  * 

E 

Tube  No. 

'•C 

c 
«< 

To  Be 
Examined. 

d 

> 

+3 

II 

B 

M 

i^ 

'M 

1 

ft 

1 

(D  3 

»o 

5| 

loM 

1 

j*> 

w 

Result. 

I... 
II... 
III... 

0.2 
0.1 
0  4 

0.2 
0.1 

... 

... 

1.0 
1.0 
1  0 

1.6 
1.8 
1  6 

1.0 
1.0 
1  0 

1.0 
1.0 
1  0 

Fixation. 

n 

Hemolysis 

IV... 
V... 
VI... 
VII... 

0.2 
0.2 

6.'2 

0.2 
02 

6i2 

1.0 
1.0 
1.0 
1  0 

1.6 
1.6 
1.8 
1  8 

1.0 
1.0 
1.0 
1  0 

1.0 
1.0 
1.0 
1  0 

Fixation. 

Hemolysis. 
<  < 

« 

VIII... 

0  2 

1  0 

1  8 

1  0 

1  0 

«  < 

IX... 
X.. 

... 

... 

... 

1.0 
1.0 

3.0 
2  0 

1.0 
1.0 

i  6 

No  hemolysis. 
Hemolysis. 

XI... 

4.0 

1.0 

No  hemolysis. 

CHAPTER  X 

EXAMINATION  OF  FLUIDS  OBTAINED  BY 
PUNCTURE 

A.— General  Characteristics  and  Chemical  Examina- 
tion 

1.  Transudates- — Transudates  are  light  yellow  with  a 
tinge  of  green  usually  transparent,  acid  in  reaction,  and 
deposit  fibrin  on  standing,  as  a  rule,  in  the  form  of  a 
moderately  gelatinous  or  membranous  clot.     The  precipi- 
tation of  the  clot  may  be  somewhat  hastened  by  the  addi- 
tion of  a  small  amount  of  blood.     This  slight  admixture 
of  blood  usually  takes  place  at  the  time  of  puncture. 

The  specific  gravity  of  transudates  is  comparatively 
low,,  and  varies  with  the  location  of  the  transudate. 
According  to  the  investigations  of  Reuss  the  specific 
gravity  of  transudates  varies  from  1,005  to  1,015.  The 
highest  specific  gravities  (to  1,015)  are  found  in  hydro- 
thorax,  the  lowest  in  hydrocephalus. 

The  albumin  contained  in  transudates  is  slight  in 
comparison  with  that  contained  in  exudates,  and  rarely 
exceeds  2.5  per  cent. 

2.  Exudates. — Exudates  show  greater  variations.    A  dis- 
tinction is  made  between  serous,  hemorrhagic,  purulent, 
and  sanious  exudates.     The  color,  transparency,  and  con- 
sistency of  these  products  of  inflammation  also  vary  cor- 
respondingly.      The  specific    gravity   is    almost  always 
above  1,018.     The  albumin  contained  rarely  sinks  below 

286 


FLUIDS   OBTAINED   BY   PUNCTURE  287 

2.5  per  cent.      However,  the  differences  in  the  specific 
gravity  and  in  the  albumin  are  not  so  constant  as  to  ren- 
der it  possible  to  differentiate  between  exudates  and  trans- 
udates  in  every  case  by  these  two  factors  alone.     Trans- 
udates  are  occasionally  found  whose  albumin  exceeds  the 
lower  limit  of  the  albumin  contained  in  exudates,  and 
rice  versa.     It  has  recently  been  claimed  that  exudates 
iffer  fromtransudates  in  that  they  contain  an  albuminoid 
body  (according  to   Umber,  seroso-mucin)  which  is  pre- 
cipitated by  acetic  acid.     The  presence  of  this  albuminoid 
substance  is  detected  by  the  fact  that  a  solution  clarified 
by  filtration  shows  a  marked  turbidity,  or  throws  down 
a  precipitate,  when  rendered  distinctly  acid  with  acetic 
acid. 

3.   Ovarian  Cysts —The  contents  of  ovarian  cysts  are 
usually  viscid  and  mucoid  in  consistency,  light  yellow 
and  occasionally  dirty  brown  or  yellowish-green  in  color! 
The  specific  gravity  varies  greatly   (between    1,005  and 
1 , 050) .     The  presence  of  peculiar  albuminoid  substances 
of   which   pseudo-mucin    (also   called    para-albumin   or 
metalbumin)  is  most  frequently  found,  is  characteristic 
the  contents  of  ovarian  cysts.     Pseudo-mucin  is  not 
precipitated  by  acetic  acid,  nitric  acid,  or  boiling,  but  is 
precipitated   by   alcohol,  and   thus    differs  widely   from 
mucin  and  albumin.     The  ordinary  varieties  of  albumin 
(albumin,  globulin)  are  present  in  varying  quantity  in 
the  contents  of  ovarian  cysts. 

Pseudo-mucin  is  detected  in  the  following  manner: 
(1)  25  cc  of  the  fluid  are  treated  with  a  few  drops  of  an 
alcoholic  solution  of  rosolic  acid,  heated  to  the  boiling- 
point,  and  treated  with  dilute  (decinormal)  sulphuric 
acid,  until  the  change  of  color  to  yellow  indicates  that  the 
fluid  is  faintly  acid.  It  is  again  heated  to  the  boiling- 
point  and  filtered.  If  the  filtrate  is  clear,  no  pseudo- 


288    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

mucin  is  present.  If  the  filtrate  is  turbid,  it  suggests 
pseudo-mucin,  but  does  not  determine  it  with  certainty, 
since  the  turbidity  may  be  due  to  albumin  which  has  not 
been  entirely  removed.  The  following  test  must  be  carried 
out  to  confirm  a  positive  reaction :  (2)  10  to  15  cc  of  the 
fluid  (depending  upon  its  specific  gravity)  are  freed  from 
albumin  by  boiling,  and  precipitated  with  three  times  the 
volume  of  95  per  cent,  alcohol.  The  flaky  precipitate  is 
collected  on  a  filter,  dried  between  filter-paper,  and  dis- 
solved in  water.  In  the  presence  of  pseudo-mucin,  an 
opalescent  solution  is  produced.  Acetic  acid  is  added, 
and  the  solution  filtered.  To  the  filtrate  one-quarter  of 
its  volume  of  25  per  cent,  hydrochloric  acid  is  added 
(4  parts  filtrate,  1  part  25  per  cent,  hydrochloric  acid). 
The  solution  is  then  heated  in  a  water-bath,  five  to  ten 
minutes  (until  it  becomes  brownish-yellow  or  brown) . 
After  it  has  cooled  it  is  neutralized  with  concentrated 
sodium  hydrate,  and  tested  with  Feliliiufs  and  Nylander^s 
tests.  If  pseudo-mucin  is  present,  both  tests  give  a  posi- 
tive result. 

4.  Hydronephrosis. — The    contents    of    hydronephroses 
usually  resemble  dilute  urine,  but  their  appearance  may 
be  altered  by  the  admixture  of  pathological  constituents 
(mucus  and  pus).     The  detection  of  both  urea  and  uric 
acid  suffices  for  the  identification  of  a  fluid  as  hydrone- 
phritic.      It  must,   however,    be   remembered  that  these 
urinary  constituents  may  be   absent  in  old,  thoroughly 
closed  cysts.     Concerning  the  detection  of  urea  and  uric 
acid,  cf.  p.  125. 

5.  Echinococcus  Cysts. — Echinococcus  fluid  is  usually 
clear,  of  low  specific  gravity,  alkaline  or  neutral  in  reac- 
tion, and  contains  considerable  sodium  chloride  and  no 
albumin,  or  only  a  very  slight  quantity.     Succinic  acid  I 
and  its  salts  are  considered  as  characteristic  constituents 


FLUIDS   OBTAINED   BY  PUNCTURE  289 

of  echinococcus  cysts,  since  they  have  frequently  been 
found  in  them  in  small  quantities.  Succinic  acid  is 
detected  in  the  following  simple  manner : 

The  fluid  is  evaporated  down  to  a  syrupy  consistency, 
acidified  with  hydrochloric  acid,  and  extracted  with  ether 
containing  alcohol.  The  ether  is  removed  by  evaporation 
in  a  water-bath,  and  the  succinic  acid  remains  as  a  crystal- 
line residue.  Microscopical  examination  reveals  hexa- 
gonal plates  or  monoclinic  prisms.  When  heated  in  a 
platinum  dish,  choking  fumes,  which  have  a  peculiar 
odor,  are  given  off.  An  echinococcus  cyst  can  be  diag- 
nosed with  absolute  certainty,  however,  only  by  micro- 
scopical examination  (detection  of  booklets  or  mem- 
brane). 

6.  Pancreatic  Cysts. — The  contents  of  pancreatic  cysts 
are  usually  hemorrhagic.     They  contain,  as  a  rule,  albu- 
min  (serum   albumin),   and    occasionally  mucin.      The 
presence  of  a  diastatic  ferment  can  usually  be  detected, 
but  is  of  little  value  in  diagnosis,  since  diastase   may 
appear  in  other  fluids  of  the  body.       The  detection  of 
trypsin  is  much  more  important,  and  is  accomplished  by 
treating  the  fluid  with  milk,  placing  it  for  some  time  in 
an  incubator)  precipitating  the  casein,  and  testing  the  fil- 
trate for  the  biuret  reaction.     A  positive  result  of  the  test 
indicates  that  the  fluid  can  digest  albumin  in  the  presence 
of  an  alkaline  reaction. 

This  proves  that  the  fluid  is  from  a  pancreatic  cyst, 
since,  as  yet,  a  ferment  capable  of  peptonizing  in  the 
presence  of  an  alkaline  reaction  has  been  detected  in  no 
other  aspirated  fluid.  This  ferment  may,  however,  be 
absent  in  old  encapsulated  cysts. 

7.  Cerebro- Spinal  Fluid. — In  healthy  persons  the  fluid 
obtained  by  lumbar  puncture  is  colorless,  clear,  and  of 
low  specific  gravity  (1,003  to  1,006).     Its  chemical  com- 


290     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

position  is  in  no  way  characteristic,  so  that  only  its  micro- 
scopical and  bacteriological  examination  are  of  diagnostic 
value. 

B.  Microscopical  Examination 

The  fluid  is  allowed  to  stand  for  some  hours  in  a  coni- 
cal glass  and  a  few  drops  of  the  precipitate  removed  with 
a  pipette  and  examined  microscopically.  If  the  quantity 
of  fluid  is  small  and  it  contains  but  little  suspended 
matter,  it  is  centrifugalized.  Unstained  smears  are  first 
examined.  Stained  smears  are  best  prepared  by  spreading 
the  sediment,  with  a  small  pipette,  in  a  thin  layer  on  a 
cover-glass,  allowing  it  to  dry  in  the  air,  and  simultane- 
ously fixing  and  staining  according  to  May  and  Gruen- 
wald  (cf.  p.  339). 

Transudates  contain  but  few  solid  constituents,  a  few 
leucocytes  in  a  state  of  fatty  degeneration,  and  isolated, 
flat  epithelial  cells.  Serous  exudates  contain,  as  a  rule, 
in  addition  to  the  fibrin  clot  and  red  blood-corpuscles 
(the  latter  usually  become  mixed  with  the  fluid  at  the 
time  of  puncture),  many  leucocytes,  and  epithelial  cells 
in  a  state  c/f  granular  or  fatty  degeneration,  which  fre- 
quently show  large  vacuoles.  In  the  presence  of  neo- 
plasms (cancer)  the  number  of  cells  with  vacuoles  is 
markedly  increased ;  they  are  in  an  advanced  stage  of  fatty 
degeneration,  and  lie  in  large  groups.  Such  groups  of 
cells  must  awaken  suspicion  of  neoplasms  if  they  are  found 
in  a  hemorrhagic  exudate.  French  authors  accredit  diag- 
nostic value  to  the  different  varieties  of  leucocytes  con- 
tained in  the  fluids.  They  have  originated  the  follow- 
ing so-called  cytological  scheme: 

1.  Excess  of  lymphocytes — i.e.,   mononuclear    leuco- 
cytes— indicates  that  the  exudate  is  tubercular. 

2.  Excess  of  polynuclear  and  eosinophilic  leucocytes 


FLUIDS   OBTAINED   BY  PUNCTURE  291 

indicates  that  the  exudate  is  infectious  but  not  tuber- 
cular. 

3.  Excess  of  endothelial  cells  indicate  that  the  fluid 
is  of  mechanical  origin  (transudate  in  cardiac,  renal,  and 
hepatic  diseases). 

This  scheme  holds  in  a  large  number  of  cases,  but, 
unfortunately,  not  in  all. 

In  the  examination  of  echinococcus  fluid  the  detection 
of  booklets  and  membrane  is  of  much  greater  diagnostic 
value  than  the  chemical  detection  of  succinic  acid.  In 
ovarian  cysts,  in  addition  to  the  red  and  white  blood- 
corpuscles,  cells  in  a  state  of  fatty  degeneration,  and 
having  vacuoles,  are  found.  Cylindrical  and  ciliated  epi- 
thelial cells,  goblet  cells,  and  colloid  concretions,  are 
characteristic  of  ovarian  cysts. 

Cerebro-spinal  fluid  is  normally  clear,  and  contains 
only  occasional  solid  constituents  (leucocytes).  In  dis- 
ease the  number  of  solid  constituents  is  very  frequently 
increased. 

C.  Bacteriological  Examination 

1.  Collection  of  Material  for  Examination 

Material  for  examination  is  obtained  by  means  of  ex- 
ploratory puncture,  or  occasionally  during  the  therapeu- 
tic measures  (operation  for  empyema,  lumbar  puncture, 
etc.). 

For  collecting  intraperitoneal  fluids  a  trocar  is  used, 
which  is  introduced,  with  the  patient  in  the  sitting  pos- 
ture, in  the  left  side  of  the  abdomen,  half-way  between 
the  symphysis  and  the  anterior  superior  spine  of  the  ilium. 
The  fluid  is  collected  in  a  sterile  flask. 

Pleuritic  effusions  are  collected  for  examination  by 
means  of  exploratory  puncture  with  a  sterile  syringe,  of 


292     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

a  capacity  of  2  to  10  cc,  and  having  a  long  (about  7  cen- 
timetres) steel  needle,  of  medium  weight.  Immediately 
before  aspiration  the  patient  must  be  examined  in  the 
position  in  which  the  puncture  is  to  be  made,  in  order  to 
determine  the  position  of  the  exudate.  The  puncture  is 
always  made  on  the  upper  border  of  the  rib,  in  widespread 
effusions  on  the  left  side,  in  the  sixth  or  seventh;  on  the 
right  side  in  the  fourth  or  fifth  intercostal  space,  between 
the  anterior  and  midaxillary  lines;  at  the  back  in  the 
eighth  or  ninth  intercostal  space. 

In  meningeal  effusions  the  material  for  examination 
is  obtained  by  means  of  lumbar  puncture,  suggested  by 
Quinclce.  There  are  two  sets  of  instruments  for  this  op- 
eration in  use.  One  suggested  by  QuincJce^  the  other  by 
Kroenig.  For  spinal  puncture  the  patient  should  be  on 
the  side,  with  the  back  curved  and  the  thighs  drawn  up 
on  the  body.  The  needle  is  introduced  between  the  fifth 
vertebra  and  the  sacrum,  a  few  millimetres  from  the  median 
line.  Quincke  recommends  the  third  or  fourth  interartic- 
ular  space  as  the  site  of  the  puncture.  The  hiatus 
sacro-lumbalis  is,  however,  better  adapted  for  diagnostic 
purposes,  since,  "owing  to  the  conical  form  of  the  lower 
portion  of  the  arachnoid  sac,  it  allows  a  natural  sedimenta- 
tion of  the  histological  and  bacteriological  substances" 
(Kroenig) .  The  evacuation,  which  must  always  be  con- 
trolled with  a  manometer,  must  be  stopped  as  soon  as  the 
pressure  sinks  below  50  millimetres.  The  fluid  is  collected 
in  sterile  test-tubes,  in  quantities  of  10  to  20  cc  per  tube. 

2.  Method  of  Examination 

1.  Microscopical  Examination.— If  the  fluid  has  a  puru- 
lent character,  a  smear  is  either  made  from  it  at  once,  or 
a  portion  is  centrifugalized,  and  the  sediment  used  for  the 
preparation  of  stained  smears.  The  smears  are  stained 


FLUIDS   OBTAINED    BY   PUNCTURE  293 

with  dilute  carbol-fuchsin  or  methylene  blue,  according  to 
Gram,  and  for  tubercle  bacilli.  For  the  detection  of  the 
latter  the  sedimentation  method  described  under  Exami- 
nation of  the -Sputum  may  be  used.  If  the  fluid  contains 
blood,  potassium  hydrate  must  be  added  before  it  has 
coagulated.  If  the  fluid  is  serous,  it  is  advisable  to  col- 
lect as  much  as  possible  of  it,  since  the  number  of  micro- 
organisms contained  is  frequently  very  small,  and  the 
possibility  of  their  detection  increases  with  the  quantity 
of  material  obtained  for  examination.  Serous  fluids  are 
allowed  to  stand  six  to  twenty- four  hours  in  the  receptacle 
in  which  they  are  collected,  in  an  ice-chest.  During  this 
time  a  clot  resembling  a  spider's  web  frequently  forms, 
which  collects  the  bacteria  present  in  the  fluid.  This  clot 
is  removed  in  toto  with  a  platinum  wire,  carefully  spread 
on  a  slide,  dried  in  the  air,  fixed,  and  stained.  If  such 
a  clot  is  not  formed,  as  large  a  portion  of  the  fluid  as 
possible  is  centrifugalized  in  the  same  centrifuge-tube. 
Stained  smears  are  made  from  the  sediment.  It  is  not 
advisable  to  fix  these  smears  in  the  flame,  but  rather  for 
three  minutes  in  alcohol  and  ether  aa.  The  method  of 
May  and  Gruenwald  yields  good  results  (cf.  p.  339). 

Jousset  has  recently  recommended  his  method  of  ino- 
scopie  (is  ivos  =  fibrin)  in  the  examination  of  serous 
fluids,  especially  for  tubercle  bacilli,  but  also  for  other 
bacteria.  After  the  clot  has  formed  it  is  separated  from 
the  fluid  by  filtration,  washed  with  distilled  water,  and 
treated  with  10  to  80  cc  of  the  following  mixture : 

Pepsin® 2.0 

Glycerini  pur., 

Acidi  hydrochlorici,  22°Baume  .     aa  10.0 

Natrii  fluorat 3.0 

Aquadest ad  1000 


294     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

This  digestive  fluid,  containing  the  clot,  is  placed  for 
two  to  three  hours  in  an  incubator  at  87°  C.  During  this 
time  the  fibrin  and  the  cell  protoplasm  are  dissolved, 
while  the  bacilli  remain  intact,  and  lose  none  of  their 
staining  characteristics.  The  fluid  is  then  centrifugalized, 
and  specimens  made  from  the  sediment.  If  no  clot  forms 
spontaneously,  Jousset  suggests  artificial  coagulation  by 
means  of  a  proper  medium  (for  example,  plasma  of  horse- 
blood). 

Frequently  the  pathogenic  bacteria  are  present  in  the 
fluids  in  such  small  numbers  that  a  number  of  specimens 
must  be  examined  before  any  bacteria  are  found.  In  the 
examination  of  cerebro-spinal  fluid  for  tubercle  bacilli, 
Slawylc  recommends  that  the  last  fluid  which  escapes  at  the 
time  of  puncture  be  examined,  since  it  contains  a  larger 
number  of  tubercle  bacilli.  If  no  micro-organisms  are 
detected  microscopically,  cultural  procedures  and,  if  neces- 
sary, animal  inoculation  must  be  resorted  to  for  their  de- 
tection. In  a  number  of  the  cases,  especially  in  old  effu- 
sions, these  methods  also  fail. 

2.  Cultural  Procedures. — The  choice  of  the  culture  media 
depends  upon  the  variety  of  micro-organisms  found  in  the 
smears.  When  the  microscopical  examination  is  nega- 
tive, various  culture  media  must  be  used.  Ordinarily, 
agar,  glycerine-agar,  serum  media,  and  blood-agar  (for 
the  cultivation  of  influenza  bacilli)  are  used.  The  culture 
medium,  contained  in  Petri  dishes,  is  inoculated  in  the 
usual  manner  with  the  material  to  be  examined.  If,  as 
is  frequently  the  case  with  serous  fluids,  no  bacteria,  or 
very  few,  are  detected  microscopically,  the  sediment  ob- 
tained by  centrifugalization  is  used  for  inoculation.  In 
the  cultural  examination  of  serous  fluids  for  tubercle 
bacilli,  80  to  50  drops  are  allowed  to  run  into  blood-serum 
or  glycerine-agar  tubes.  The  excess  of  the  fluid  is  allowed 


FLUIDS   OBTAINED   BY   PUNCTURE  295 

to  evaporate  in  the  incubator  at  37°  C. ,  and  the  applica- 
tion of  rubber  caps  to  prevent  drying  of  the  culture  media 
is  postponed  until  but  little  fluid  remains. 

3.  Animal  Inoculation. — Animal  inoculation  serves  the 
purpose  first  of  detecting  micro-organisms  in  the  fluids, 
and  second  of  identifying  the  bacteria  found  in  the  smears, 
or  by  cultural  methods.  The  choice  of  the  test-animals  and 
the  method  of  inoculation  must  be  suited  to  the  pathogenic 
bacteria  whose  presence  is  suspected,  or  whose  identifica- 
tion is  desired.  For  example,  white  mice  are  used  for  the 
identification  or  detection  of  streptococci  or  pneumococci, 
and  guinea-pigs  for  tubercle  bacilli  (cf.  Examination  of 
the  Sputum). 

In  examining  for  tubercle  bacilli,  animal  inoculation 
will  succeed  more  frequently  than  microscopical  or  cul- 
tural methods.  In  the  examination  of  serous  fluids, 
either  the  above-mentioned  coagulum  is  used  for  inocula- 
tion, or  a  considerable  quantity  of  the  exudate — at  least 
4  cc — is  injected  into  the  animal.  In  purulent  exudates 
the  sediment  obtained  by  centrifugalization  is  used. 

3.  The  Most  Important  Bacteriological  Findings 

1.  Peritoneal  Exudates. — The  bacteriological  findings  in 
acute  peritonitis  depend  principally  upon  the  locality  from 
which  the  inflammation  extends.  Mixed  infections  are 
very  frequent.  In  peritonitis  of  intestinal  origin,  princi- 
pally bacilli  belonging  to  the  group  of  Bacterium  coli  are 
found,  together,  as  a  rule,  with  other  micro-organisms  of 
the  intestinal  flora,  as  staphylococci,  Proteus  vulgaris, 
Bacillus  pyocyaneus,  etc.  Peritonitis  extending  from  the 
female  genital  organs  is  most  frequently  caused  by  gono- 
cocci ;  puerperal  peritonitis  by  streptococci  and  other  pyo- 
genic  bacteria.  In  peritonitis  in  which  the  infection  has 
reached  the  peritoneum  by  means  of  the  circulation,  strep- 


296    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

tococci  and  pneumococci  have  been  most  frequently  found; 
in  peritonitis  following  operations,  streptococci.  Typhoid 
bacilli,  actinomyces,  pseudo-diphtheria  bacilli,  and  bacilli 
resembling  tetanus  bacilli  have  also  been  found  in  peri- 
toneal exudates.  Finally,  the  tubercle  bacillus  is  of  great 
importance  as  the  exciting  cause  of  chronic  peritonitis. 

2.  Pleuritic  Exudates. — Pleuritic  exudates  are  also  of 
varying  origin.  Tubercle  bacilli,  as  well  as  all  the  pyo- 
genic  micro-organisms,  may  be  the  exciting  cause  of  pleu- 
ritic effusion.  Serous  exudates  are  by  far  most  frequently 
tubercular.  A  negative  microscopical  and  cultural  exami- 
nation should  arouse  strong  suspicion  that  they  are  tuber- 
cular. The  inoculation  of  guinea-pigs  must  always  be 
used  in  the  diagnosis  of  such  cases. 

In  purulent  exudates  streptococci  are  most  frequently 
found;  next,  pneumococci,  staphylococci,  and  tubercle 
bacilli;  more  rarely,  influenza  bacilli  and  Micrococcus 
tetragenus.  Typhoid  bacilli  have  been  detected  in  pleuritic 
effusions  in  the  course  of  typhoid  fever.  Pleuritic  effu- 
sions accompanying  pneumonia  frequently  contain  pneu- 
mococci, either  alone  or  with  staphylococci. 

Sanious  exudates  contain,  in  addition  to  the  pyogenic 
cocci,  bacteria  of  decomposition  and  anaerobic  varieties. 

8.  Meningitic  Effusions. — Normal  cerebro-spinal  fluid  is 
clear,  free  from  bacteria,  and  contains  only  occasional 
lymphocytes  and  epithelial  cells.  The  pathological  effu- 
sion is  colorless  and  clear  in  the  cerebral  cedema  of  chlo- 
rosis, uraemia,  cerebral  tumors,  and  in  serous  meningitis 
(Kroenig) . 

In  tubercular  meningitis  the  fluid  is  also  usually  clear, 
but  occasionally  somewhat  opalescent,  and  frequently  con- 
tains many  leucocytes.  In  the  early  stages  of  the  disease 
the  poly  nuclear  leucocytes  seem  to  prevail,  and  in  the 
later  stages  the  mononuclear.  In  acute  non-tubercular 


FLUIDS   OBTAINED   BY  PUNCTURE  297 

meningitis  the  character  of  the  fluid  varies  according  to 
the  intensity  of  the  process,  even  when  the  cause  is  the 
same;  it  may  be  serous,  fibrinous,  fibrino-purulent,  or 
purulent. 

The  Diplococcus  intracellularis  meningitidis  "  Weich- 
selbaum"  and  the  Diplococcus  pneumonia  have  been 
detected  as  the  exciting  cause  of  acute,  primary,  epide- 
mic, and  sporadic  cerebro-spinal  meningitis. 

Concerning  the  Diplococcus  pneumonice,  cf .  p.  48. 

The  Diplococcus  intracellularis  meningitidis  appears 
usually  as  a  diplococcus  or  tetracoccus ;  the  cocci  are  some- 
what flattened  on  the  inner  side,  and  have  therefore  a 
hemispherical  or  coffee-bean  form.  They  often  vary  con- 
siderably in  their  size  and  staining  qualities,  so  that  in 
the  same  smear  smaller  and  larger  feebly  stained  cocci 
(degeneration  forms)  are  found  beside  the  normal  cocci. 
They  resemble  gonococci  in  form  and  arrangement,  but 
are  larger.  Like  gonococci,  they  frequently  lie  in  the  exu- 
date  in  groups  within  the  pus  corpuscles.  Their  staining 
characteristics  also  agree  with  those  of  gonococci;  they 
stain  easily  with  dilute  aniline  dyes,  and  are  decolorized 
by  Gram. 

4.  Cultural  Behavior.  —  The  Diplococcus  "Weichsel- 
fiaum"  grows  best  at  a  temperature  of  86°  to  87°  C.  Upon 
agar,  gray  to  grayish-white  colonies  are  formed  within 
twenty-four  hours,  which  are  1  to  2  millimetres  in  diam- 
eter, have  smooth  or  wavy  margins,  and  are,  when  exam- 
ined against  the  light,  transparent  and  yellowish. 

Upon  serum  plates  the  growth  is  more  luxuriant;  the 
grayish-yellow  colonies  are  moistly  glistening  and  viscid, 
like  those  of  the  diplococcus  of  Priedlaender. 

Their  cultivation  from  meningitic  exudates  succeeds 
best  on  -serum,  but  it  occasionally  fails  even  on  this 
medium. 


298     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Animal  inoculation  is  not  used  for  diagnostic  purposes. 
White  mice  die  in  twenty-four  to  forty-eight  hours  follow- 
ing the  intrapleural  injection  of  a  comparatively  large 
quantity  of  a  culture.  Post  mortem,  the  diplococci  are 
found  both  within  and  without  the  cells  in  the  pleuritic 
or  peritoneal  exudate. 

Pneumo-,  staphylo-,  and  streptococci,  influenza  bacilli, 
the  diplobacilli  of  Friedlaender,  Bacterium  coli,  and  plague 
bacilli  have  been  reported  as  the  exciting  cause  of  cerebro- 
spinal  meningitis  secondary  to  infectious  disease. 


CHAPTER  XI 

BACTERIOLOGICAL  EXAMINATION  OF 
DISEASES  OF  THE  SKIN 

Purulent  Affections  of  the  Skin 

Material  for  examination  is,  as  a  rule,  obtained  by 
puncture  with  a  sterile  needle,  or  by  incision.  If  the  bac- 
teriological examination  happens  to  be  undertaken  at  the 
time  of  an  operation,  especial  care  must  be  taken  that  the 
material  for  examination  does  not  come  in  contact  with 
disinfectants. 

The  material  is  usually  examined  microscopically  and 
by  means  of  cultures.  Animal  inoculation  is  used  in  case 
these  two  methods  fail,  or  to  identify  bacteria  which  have 
been  cultivated. 

As  the  exciting  cause  of  furuncular  processes,  Staphy- 
lococcus  aureus  or  albus  can  almost  always  be  detected 
microscopically,  and  by  cultivation  upon  the  usual  culture 
media.  In  the  pus  of  panaris  (whitlow),  in  addition 
to  staphylococci,  streptococci,  and  more  rarely  Bacterium 
coti,  may  be  found.  In  acute  abscesses  and  phlegmona, 
in  addition  to  the  above-mentioned  pyogenic  bacteria, 
pneumococci,  typhoid  bacilli,  etc.,  may  be  found.  In 
large  abscesses  the  detection  of  micro-organisms  frequently 
fails  in  pus  taken  from  their  centre,  while  in  the  periph- 
ery, in  the  so-called  abscess  membrane,  their  detection 
is  easy.  In  the  so-called  cold  abscesses  the  bacteria  can, 
as  a  rule,  be  detected  neither  microscopically  nor  by  cul- 

299 


300     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

tural  means.  The  presence  of  tubercle  bacilli  in  them 
can  also,  as  a  rule,  be  detected  only  by  inoculating  guinea- 
pigs  with  the  pus. 

In  the  pus  of  gas  phlegmona,  bacilli  belonging  to  the 
group  of  Bacterium  coli  and  Bacterium  lactis  aerogenes, 
as  well  as  anaerobic  bacteria  (B.  empliysematosus),  may 
be  found,  in  addition  to  the  ordinary  pyogenic  bacteria. 

In  multiple  abscesses  developing  in  the  skin  and  mus- 
cles, in  the  course  of  glanders,  it  is,  as  a  rule,  impossible  to 
detect  the  Bacillus  mallei  microscopically.  In  suspected 
cases  cultures  are  planted  upon  glycerine-agar  and  potato, 
and  animal  inoculation  is  used.  The  potato  cultures  are 
very  characteristic.  After  two  days,  a  honey-yellow  coat- 
ing can  be  seen,  which,  after  a  week,  is  brownish-red,  and 
surrounded  by  a  slightly  greenish,  shimmering  zone. 
Upon  glycerine-agar  transparent  grayish  colonies  are  seen. 

Bacilli  mallei  are  small,  slim,  slightly  curved,  non- 
motile  rods,  about  the  size  of  tubercle  bacilli.  They  do 
not  stain  with  dilute  dyes,  but  best  with  Loeffler's  alkaline 
methylene  blue. 

Male  guinea-pigs  are  used  as  test  animals.  The  sus- 
pected material  is  injected  into  the  peritoneal  cavity  in 
the  median  line  above  the  bladder.  After  two  to  three 
days  the  testicles  become  swollen,  which  is  a  character- 
istic symptom  of  successful  transmission  of  glanders. 
Potato  cultures  are  inoculated  from  the  diseased  testicles. 

Anthrax  carbuncle,  the  so-called  malignant  pustule,  is 
due  to  infection  with  anthrax  bacilli.  Lymph  obtained 
from  the  deep  portion  of  the  suspected  pustule  is  used  as 
material  for  examination.  The  serous  contents  of  the 
pustule  are  free  from  bacilli,  since  the  latter  lie  about  the 
papillse,  in  the  external  portion  of  the  corium.  Speci- 
mens are  stained  with  dilute  methylene  blue,  according  to 
Gram,  and  by  one  of  the  methods  which  serve  to  demon- 


SKIN   DISEASES  301 

strate  capsules.  Microscopical  examination,  however, 
yields  positive  results  only  for  a  short  time  following  the 
formation  of  the  carbuncle;  later,  the  bacteria  can  be  de- 
tected only  by  means  of  cultures  or  animal  inoculation, 
which  may  also  fail.  Gelatine  and  agar  plates  are  inoc- 
ulated with  the  lymph.  After  twenty-four  hours'  growth, 
characteristic  colonies  of  anthrax  bacilli  are  seen.  Staphy- 
lococci  also  frequently  develop  along  with  them.  Further, 
white  mice  or  guinea-pigs  are  inoculated  by  implanting 
the  material  to  be  examined  in  a  pocket  under  the  skin 
above  the  base  of  the  tail.  Bacilli  which  develop  in  the 
cultures  are  also  identified  by  animal  inoculation. 

Anthrax  bacilli  (Plate  XV,  Fig.  Z)  are  clear,  cylindri- 
cal, non-motile  rods,  with  rounded  ends,  and  of  varying 
length ;  they  appear  much  larger  in  cultures  than  in  the 
animal  organism.  They  stain  easily  with  dilute  aniline 
dyes,  and  according  to  Gram.  In  stained  smears  the 
bacilli  usually  have  a  slight  bulbous  enlargement  at  their 
ends,  and,  at  the  same  time,  a  slight  concavity,  so  that 
when  two  bacilli  lie  end  to  end,  a  small  hole  is  formed 
between  the  points  of  contact  (bamboo- form).  In  the 
Gram  specimen  they  are  often  unevenly  stained,  and  ap- 
pear granular.  In  specimens  obtained  from  animal  organ- 
isms anthrax  bacilli  possess  a  mucoid  covering,  the  so- 
called  capsule,  which  may  be  demonstrated  by  special 
staining  methods  (cf.  p<  385). 

Anthrax  bacilli  form  centrally  placed  spores  in  the 
presence  of  free  O,  and  at  temperatures  above  18°  C. 

Anthrax  bacilli  grow  upon  all  the  usual  culture  media. 
Upon  agar  and  gelatine  they  develop  very  characteristic 
colonies.  On  examination  with  the  low  power,  numerous 
spiral  branches  are  seen  extending  from  a  centre  composed 
of  a  non-transparent  whorl  of  threads,  which  give  the 
colony  the  appearance  of  a  tangle  of  hair.  Bouillon  is 


302    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

not  clouded  in  toto,  but  a  sediment  is  formed.  In  gela- 
tine stab  cultures  the  bacilli  grow  along  the  stab  canal  and 
form  delicate  branches  from  it.  Gelatine  is  liquefied; 
milk  is  coagulated. 

White  mice  and  guinea-pigs  are  used  as  test  animals 
for  diagnostic  purposes.  The  animals  die  of  anthrax 
septicaemia  one  to  three  days  following  subcutaneous  inoc- 
ulation. Post  mortem,  the  spleen  is  found  greatly  en- 
larged. Bacilli  are  found  only  in  small  number  in  the 
heart's  blood,  but  in  great  number  in  the  capillaries  of 
all  the  viscera,  especially  in  the  spleen  and  liver,  and 
show,  in  microscopical  specimens,  the  characteristic  cap- 
sules. 

Bacilli  of  malignant  oedema  must  be  considered  in 
making  a  differential  diagnosis.  These  are  motile,  have 
no  capsule,  and  are  absolutely  anaerobic.  Anthrax  bacilli 
are  distinguished  from  the  saprophytes  which  form  simi- 
lar colonies  (potato,  and  hay  bacilli)  by  their  typical 
morphological  characteristics,  and  especially  by  the  fact 
that  they  are  pathogenic. 

The  Detection  of  Tetanus  Bacilli  (Plate  XVI,  Fig.  a)  in 
the  Secretion  of  Infected  Wounds. — Tetanus  bacilli  are  pres- 
ent in  such  small  number  in  the  secretion  of  the  wounds 
that  they  cannot  be  detected  microscopically.  Cultural 
procedures  also  often  yield  a_negative  result.  Animal  in- 
oculation is  much  more  frequently  successful.  For  this 
purpose,  secretion  from  the  wound,  granulation  tissue,  or 
any  foreign  body  found  in  the  wound,  is  used,  mice  and 
guinea-pigs  being  inoculated  in  a  pocket  under  the  skin 
of  the  thigh.  If  the  inoculated  animals  show  no  signs  of 
tetanus  within  five  days,  the  result  should  be  considered 
negative.  A  positive  result  of  animal  inoculation  is  suffi- 
cient for  diagnosis,  it  being  unnecessary  to  grow  the 
bacilli  in  pure  culture. 


SKIN   DISEASES  303 

Tetanus  bacilli  are  slightly  motile,  slim  rods,  which  in 
smears  made  from  pure  cultures,  lie  singly  or  arranged  in 
threads  of  varying  length.  At  room-temperature  after 
eight  to  ten  days,  and  at  incubator-temperature  after 
twenty- four  to  thirty  hours,  they  form  spores  at  one  end, 
which  give  them  the  appearance  of  a  drum-stick.  Tetanus 
bacilli  stain  easily  with  dilute  aniline  dyes,  and  according 
to  Gram. 

Cultural  Behavior. — Tetanus  bacilli  are  anaerobic.  They 
grow  in  the  absence  of  air  on  all  the  usual  culture  media, 
especially  well  if  grape-sugar  (2  per  cent.)  is  added.  In 
symbiosis,  with  aerobic  bacteria,  they  grow  even  in  the 
presence  of  oxygen. 

Cultivation  of  Pure  Cultures  According  to  Kitasato. — The 
material  to  be  examined  is  planted  upon  agar  tubes,  which 
are  placed  for  one  to  two  days  in  the  incubator,  after  which 
time  tetanus  bacilli  having  spores  are  present  among  the 
other  bacteria.  The  mixed  culture  is  now  heated  in  a 
water-bath  at  80°  C.  for  about  one  hour,  by  which  the 
other  bacteria  are  killed,  while  the  resistant  tetanus  spores 
remain  capable  of  development.  From  these,  anaerobic 
cultures  are  made  in  the  usual  manner  (cf.  p.  359). 

After  five  days7  growth  on  gelatine,  small  colonies  with 
radiating  branches  have  developed.  Gelatine  is  liquefied. 
They  develop  much  more  rapidly  upon  agar.  When  ex- 
amined with  the  low  power,  the  delicate  colonies  appear 
as  a  maze  of  fine  threads. 

Animal  Inoculation. — Mice  and  guinea-pigs  are  the  most 
sensitive  test  animals,  and  are  inoculated  by  means  of  a 
piece  of  wood,  or  the  like,  which  is  impregnated  with  the 
material  to  be  examined  and  introduced,  through  a  nick, 
under  the  skin.  The  first  symptoms  of  tetanus  appear  in 
the  muscles  near  the  site  of  inoculation.  The  animals  die 
with  their  hind  legs  stretched  out.  Tetanus  bacilli  can 


304    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

be  detected  microscopically  and  by  cultural  methods  only 
at  the  site  of  inoculation. 

The  Detection  of  the  Bacillus  of  Soft  Chancre  (ulcus 
mollej,  discovered  by  Ducrey,  is  occasionally  of  practical 
value. 

In  smears  made  from  the  secretion  of  fresh  ulcers  and 
stained  with  Loeffler's  methylene  blue,  borax-methylene 
blue,  or  polychrom-methylene  blue,  short,  thick  bacilli, 
which  have  rounded  ends  and  frequently  show  polar  stain- 
ing, and  which  lie  in  groups,  pairs,  or  singly,  both  within 
and  without  the  cells,  appear  in  addition  to  other  micro- 
organisms. They  are  decolorized  by  Gram.  The  picture 
which  they  present  in  sections,  made  from  the  periphery 
of  the  excised  soft  chancre,  is  characteristic.  The  bacilli 
frequently  lie  in  long  parallel  chains,  always  outside  of 
the  cells,  in  the  lymphatic  spaces  of  the  tissue,  and  every- 
where a  little  beyond  the  border  of  the  necrotic  and  within 
the  living  tissue.  (Concerning  the  staining  of  sections, 
cf.  p.  343.) 

Bacilli  of  soft  chancre  do  not  grow  upon  the  usual  cul- 
ture media.  They  may  occasionally  be  cultivated  upon 
blood-agar  (2  parts  liquefied  agar,  which  has  been  cooled 
to  40°  to  50°  C.,  and  1  part  rabbit  blood  and  non-coagu- 
lated blood  serum  from  the  pus  of  the  ulcer  before  it  has 
ruptured  through  the  skin  covering  it,  and  from  inocula- 
tion chancres.  After  forty-eight  hours'  growth  at  37°  C., 
dark  gray,  glistening  round  colonies  the  size  of  a  pin's 
head  have  developed,  which  may  be  shifted  about  upon,  or 
lifted  bodily  from,  the  surface  of  the  culture  medium,  with 
the  platinum  needle.  The  colonies  are  composed  of  poly- 
morphous rods,  which  frequently  lie  in  rows,  and  are  seen 
to  be  non-motile  when  examined  in  a  hanging-drop. 

Transplantation  on  the  human  skin  is  used  for  the  de- 
tection of  Ducretfs  bacillus.  The  side  of  the  abdomen  of 


SKIN   DISEASES  305 

the  patient  himself  is  used  as  the  site  of  inoculation.  The 
skin  is  scarified  in  several  spots,  and  the  secretion  from 
the  ulcer  to  be  examined  is  rubbed  in.  After  two  to  four 
days,  secondary  chancres  develop,  in  whose  secretion  the 
bacilli  are  usually  found  in  great  numbers. 

Tuberculosis  of  the  Skin 

Bacterioscopy  has  but  little  diagnostic  value  as  regards 
tuberculosis  of  the  skin,  since,  as  in  other  chronic  tuber- 
cular processes,  the  bacilli  are  usually  present  in  such 
small  numbers  that  the  attempt  to  detect  them  micro- 
scopically often  fails.  They  are  most  likely  to  be  found 
in  smears  made  from  the  secretion  of  tubercular  ulcers, 
but  their  detection  in  such  cases  does  not  determine  with 
certainty  that  they  are  the  exciting  cause  of  the  disease, 
since  tubercle  bacilli  may  become  located  upon  ulcerating 
surfaces  without  having  an  etiologic  bearing  upon  the  dis- 
ease. Further,  it  must  be  remembered  infoaking  a  differ- 
ential diagnosis  that  other  acid-fast  bacilli  are  frequently 
found  on  the  skin. 

In  tuberculosis  cutis  verrucosa,  occasional  tubercle 
bacilli  are  found  in  sections. 

The  bacilli,  in  skin  affected  by  tuberculosis,  are  more 
likely  to  be  detected  by  means  of  animal  inoculation 
(subcutaneous  inoculation  of  guinea-pigs)  than  by  micro- 
scopical examination. 

Diseases  of  the  Skin  Excited  by  Hyphomycetes 
(Dermatomycosis) 

Collection  of  Material  for  Examination 

For  the  collection  of  epidermal  scales,  the  skin  is 
either  scraped  with  a  dull,  slightly  moistened  scalpel,  or 


306    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

according  to  Unna,  a  piece  of  zinc  oxide  plaster  or  ordi- 
nary surgeon's  plaster  is  laid  upon  the  skin  and  pressed 
for  a  few  minutes  with  the  warm  hand,  then  lifted,  the 
scales  which  stick  to  it  loosened  with  benzine,  and  freed 
with  HC1  alcohol  from  the  zinc  oxide  which  clings  to 
them.  Before  further  examination,  the  scales  are  placed 
in  water,  in  which  they  become  swollen. 

Hairs  are  obtained  for  examination  by  epilation. 

Small  particles  are  scraped  from  the  nails. 

Microscopical  Examination 

The  examination  of  unstained  specimens  very  fre- 
quently suffices  for  diagnostic  purposes.  The  material  to 
be  examined  is  placed  upon  a  slide,  and  either  rubbed 
with  a  40  per  cent,  solution  of  potassium  carbonate  or  a 
10  to  15  per  cent,  solution  of  potassium  hydrate,  or  crushed 
between  two  slides,  and,  after  slight  warming  over  the 
flame,  is  covered,  with  a  cover-glass  and  examined  with 
the  medium  power  (about  800).  The  oil  immersion  is 
used  for  the  detection  of  the  parasite  of  erythrasma. 

Stained  specimens  are  examined  principally  when  the 
fungi  are  present  in  such  small  numbers  that  they  escape 
detection  in  unstained  specimens.  Of  the  numerous  stain- 
ing methods  which  have  been  recommended,  Plcmtli's 
modification  of  Bizzozero's  method,  and  WaelscWs  method 
(cf.  p.  887)  should  be  mentioned. 

Hair  must  be  freed  from  fat  by  several  hours'  im- 
mersion in  a  mixture  of  alcohol  and  ether  before  it  is 
stained. 

Cultural  Procedures. — The  most  favorable  culture  media 
are  grape-sugar,  glycerine-  and  maltose-agar,  SabouraucTs 
milieu  d'epreuve  (maltose,  4.0;  peptone,  2.0;  agar-agar, 
1.5;  aqua  dest.,  100.0),  and  wort-agar.  In  cultivating 
fungi  from  the  horny  layer  of  the  skin,  and  from  the  hair 


SKIN   DISEASES  307 

and  nails,  KrdVs  method  may  be  used.  As  much  material 
as  possible  is  lightly  rubbed,  in  a  porcelain  dish,  with  cal- 
cined infusorial  earth ;  liquefied  agar  which  has  been  cooled 
to  40°  C.  is  inoculated  with  two  to  three  loops  of  the  in- 
fected earth  and  poured  into  plates.  Dilutions  may  be 
made  in  the  usual  manner.  After  two  to  three  days' 
growth  the  plates  are  examined  with  the  low  power,  and 
the  suspicious  looking  colonies  removed  and  grown  in  pure 
culture.  According  to  Sabouraud,  the  young  cultures  are 
transplanted  upon  the  surface  of  congealed  maltose-agar 
contained  in  100  cc  Erlenmeyer  flasks.  The  layer  of  agar 
should  be  1.5  centimetres  thick.  The  flasks  remain  open 
in  the  incubator. 

W.  Scliolz  recommends  the  method  used  in  the  derma- 
tological  clinic  in  Breslau,  which  is  especially  suited  to 
the  cultivation  of  favus  fungi  from  the  hair. 

The  hair  and  scales  are  freed  from  fat  by  being  placed 
for  a  few  minutes  in  ether,  washed  with  water,  placed  for 
one  to  two  minutes  in  a  1  per  cent,  solution  of  silver,  in 
order  to  kill  the  micro-organisms  clinging  to  their  surface, 
placed  for  a  short  time  in  sterile  water  and  physiological 
salt  solution,  and  again  washed  with  water.  The  material 
to  be  examined  (the  hairs  are  cut  into  small  particles)  is 
distributed  over  the  surface  of  suitable  culture  media. 
Plautli l  recommends  "cultivation  in  situ"  as  a  diagnostic 
method,  especially  for  trichophyta  which  grow  at  room- 
temperature,  but  also  for  the  favus  fungus,  which  develops 
only  at  higher  temperatures. 

Cultivation  in  Situ  at  Room- Temperature. — Several  (three 
to  four)  hairs  and  scales  are,  without  previous  preparation 
and  without  treatment  of  the  lesion,  placed  upon  a  sterile 
slide.  They  are  crushed  with  a  second  sterile  slide  in 

1  Zentralblatt  f.  Bakt.  u.  Parasitenkunde,  1902,  Bd.  31,  No.  5. 


308    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

order  to  spread  them  and  make  them  sufficiently  trans- 
parent for  microscopical  examination.  They  are  then 
covered  with  a  cover-glass,  which  is  fastened  at  opposite 
sides  with  a  drop  of  wax.  The  slide  is  now  placed  in  a 
flat,  moist  chamber.  This  consists  of  a  plate  upon  which 
is  a  glass  dish,  on  which  the  slide  is  placed,  and  a  glass 
bell,  12  centimetres  in  diameter  and  7  centimetres  in 
height.  The  interior  of  the  bell  is  draped  with  filter-paper, 
which  is  fastened  with  drops  of  wax,  and  has  an  opening 
in  the  centre,  through  which  the  culture  may  be  examined 
without  removing  the  bell.  After  the  slide  and  bell  are 
in  position,  the  plate  is  filled  with  water.  Care  must  be 
taken  that  no  water  touches  the  culture. 

If  it  is  desired  to  transplant  upon  ordinary  culture 
media,  a  small  piece  is  cut  from  the  edge  of  the  scale, 
after  the  fungi  are  well  developed,  and  transplanted  upon 
maltose-agar,  or  used  for  making  plate  cultures  according 
to  KrdVs  method. 

In  cultivating  in  an  incubator,  in  order  to  protect  the 
cover-glass  from  condensation  water,  it  is  covered  with  a 
bridge  of  moist  filter-paper  _  "1  ,  which  is  fas- 

tened at  the  ends  with  wax  and  freshly  moistened  every 
morning. 

The  development  of  mycelium  which  in  attempts  at 
cultivation  at  room-temperature  appears  in  the  first  two 
to  three  days,  and  that  which  spreads  from  the  edge  of  the 
cover-glass  and  not  from  the  hairs  and  scales,  are  to  be 
considered  as  due  to  contamination.  In  cultivating  in 
the  incubator,  contamination  with  fungi  is  easily  recog- 
nized, since  fructification  takes  place  rapidly. 

At  room-temperature,  trichophyta  develop  from  the 
sixth  to  eleventh  day  onward.  By  cultivation  in  an  incu- 
bator at  85°  C.  trichophytosis  and  favus  can  be  diagnosed 
in  this  manner  after  only  forty-eight  hours. 


SKIN   DISEASES 
Favus  (Fig.  39) 


309 


The  exciting  cause  of  favus  is  the  Achorion  ScJioen- 
leinii.     Its  detection  is  easy  as  soon  as  the  characteristic 


FIG.  39. — Favus  Fungi,  after  Lassar. 

crusts,  the  scutula,  are  present.     These  appear  as  compact, 
sulphur-yellow,    cup-shaped    bodies,    which    are  usually 


310    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY   ! 

pierced  by  a  hair  and  embedded  in  the  skin.  They  are 
isolated  by  piercing  the  horny  layer,  which  at  first  covers 
them,  and  prizing  them  out  of  the  skin.  If  the  scutula 
do  not  appear  distinctly,  they  can  be  made  distinct  by 
moistening  the  skin  with  alcohol.  The  specimens  are  ex- 
amined unstained.  The  scutulum  is  seen  to  be  composed 
of  a  finely  granular  mass,  at  the  centre  of  which  short, 
double-contoured,  oval,  round,  or  rectangular  spores  lie 
close  together,  and  at  the  periphery  of  which  radiating 
threads  of  mycelium  are  seen.  These  appear  as  threads 
of  varying  width,  with  many  septa,  often  bifurcating  and 
having  bulbous  ends.  They  also  bud  laterally  and  cut  off 
the  lateral  hyphae  almost  at  a  right  angle. 

The  second  important  seat  of  favus  fungi  is  the  hair. 
Here,  also,  they  may  be  clearly  seen  in  unstained  speci- 
mens. Longitudinal  chains  of  mycelium,  which  are  com- 
posed principally  of  rectangular  members,  are  formed. 

The  fungi  develop  within  the  sheath  of  the  root  and  in 
the  hair  itself;  principally  between  the  cuticula  and  the 
cortex,  but  also  entering  the  cortex,  as  a  rule,  without 
splitting  the  hair. 

The  detection  of  the  fungi  in  epidermal  scales,  in  which 
they  are  usually  present  in  but  small  numbers,  is  more 
difficult.  They  cannot,  as  a  rule,  be  discovered  in  un- 
stained specimens.  Bizzozero''s  staining  method  (cf.  p. 
837)  is  best  used  for  their  detection. 

The  examination  of  the  nails  is  also  made  by  means 
of  stained  specimens.  Threads  of  mycelium  with  spores 
are  usually  found.  The  favorite  seat  of  the  mycelial 
threads  is  between  the  bed  and  the  lamina  of  the  nail. 

Cultural  Procedures. — Favus  fungi  grow  best  at  85°  C. 
upon  culture  media  rich  in  nitrogen.  After  eight  days 
the  colonies  are  about  the  size  of  a  pin's  head,  and  after 
two  to  three  weeks  they  are  fully  developed.  The  micro- 


SKIN   DISEASES  311 

scopical  appearance  of  the  cultures  varies  greatly,  depend- 
ing upon  different  factors,  as  the  culture  media,  differences 
in  temperature,  age  of  the  culture,  etc.  Plauth  distin- 
guishes between  two  main  types:  (1)  The  waxy  type; 
yellowish  spots  of  waxy  consistency,  which  have  radial 
folds  and  raised  centres,  and  usually  no  air-mycelium, 
though  they  occasionally  form  a  short  down.  (2)  The 
downy  type;  white  discs  covered  with  a  thick  down,  with 
irregular  raised  centres.  The  color  varies;  they  may  be 
snow-white,  reddish,  or  yellow. 

Animal  Inoculation. — Gray  mice  are  used,  and  contract 
favus  when  the  material  is  rubbed  into  the  skin  a£  the 
base  of  the  tail.  A  negative  result  of  the  test  does  not 
exclude  favus,  since  not  all  favus  fungi  coming  from  man 
are  pathogenic  for  mice. 

Trichophytosis 

Under  the  name  of  trichophytosis  are  included  those 
diseases  of  the  skin  which  are  caused  by  fungi  belonging 
to  the  group  of  the  trichophyton.  In  spite  of  the  numer- 
ous workers  who  have  studied  the  etiology  of  these  dis- 
eases, it  has  not  as  yet  been  definitely  settled  whether  the 
different  clinical  manifestations  of  trichophytosis  are 
caused  by  one  and  the  same  micro-organism,  remarkable 
for  its  pleomorphism,  or  whether  there  are  a  variety  of  true 
trichophyta,  to  which  the  different  clinical  pictures  owe 
their  peculiarities. 

ScibouraudJ-  especially,  supports  the  latter  view.  He 
separates  from  the  true  trichophytosis  a  form  of  Tinea 
tonsurans,  the  so-called  microsporia,  which  is  caused 
by  a  small-spored  fungus,  the  Microsporon  Audouini 

1See  ' 'Regional Dermatology,"  by Sabouraud.  Rebman  Com- 
pany, New  York. 


312    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

(Gruby),  a  variety  of  fungus  which,  according  to  his  in- 
vestigations, is  entirely  distinct  from  those  exciting  other 
forms  of  trichophytosis. 

Microsporia. — Only  the  hairs  are  examined.  The  hairs, 
which  protrude  but  slightly,  break  off  when  removed 
shortly  above  the  surface  of  the  scalp,  the  roots  remaining 
in  the  matrix.  They  have  a  silvery-gray  lustre,  which 
examination  with  a  magnifying-glass  shows  is  due  to  a 
sheath  surrounding  the  hair.  Microscopical  examination 
reveals  that  the  sheath  is  almost  entirely  composed  of 
small,  closely  placed  ectospores.  Within  the  air  threads 
of  mycelium,  with  peculiar  gnarly,  short  branches,  are 
seen.  Cultivation  is  not  necessary  for  diagnostic  purposes, 
since  the  microscopical  detection  of  the  fungi  in  the  hair 
is  easy.  Other  forms  of  Tinea  tonsurans  are,  according 
to  Safiouraud,  due  to  a  large-spored  fungus.  The  fungi 
appear  within  the  hairs — which  are  thick,  break  off  close 
to  the  scalp,  and  are  difficult  to  remove — in  the  form  of 
large  round,  somewhat  irregular,  distinctly  double-con- 
toured spores,  which  form  long  rosaries. 

Tinea  Sycosis  (Fig.  40) 

In  the  superficial  form  of  Tinea  sycosis  the  detection 
of  the  fungi  is  usually  easy  in  the  hair  at  the  border  of 
the  rings.  The  spores  lie,  as  a  rule,  about  the  follicles, 
the  mycelium  longitudinally  within  the  inner  sheath  of 
the  root,  but,  also  penetrating  the  substance  of  the  hair 
itself. 

In  the  deeper  form  of  sycosis  (Sycosis  parasitica)  the 
microscopical  detection  of  the  fungus  is  more  difficult. 
Its  detection  is  easy,  however,  if  cultures  are  made  from 
the  purulent  secretion  taken  from  the  deepest  portion  of 
the  lesion. 


SKIN   DISEASES 


313 


In  order  to  tell  which  of  a  number  of  plucked  hairs 
contain  fungi,  the  hairs  are  moistened  with  chloroform; 
after  the  chloroform  has  evaporated  the  hairs  containing 


FIG.  40.— Hair  in  Sycosis. 

fungi  become  chalky  white.     If  the  hairs  are  moistened 
with  oil  they  regain  their  normal  color. 

Tinea  Circinata  (Fig.  41) 

Trichophyta  Circumscripta  and  Disseminata.1 — The  fungi 
appear  in  the  epidermal  scales  as  long,  moderately  branch- 
ing threads,  which  give  off  but  few  conidia.  They  are, 

1  See  ' '  Dermochromes. ' '    Kebman  Company,  New  York. 


314    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

however,  especially  in  Trichophyta  disseminata,  as  a  rule, 
so  isolated  that  it  is  difficult  to  find  them  even  in  stained 
specimens. 

Plauth  recommends  his  method  of  cultivation  in  situ, 
as  an  aid  in  the  diagnosis  of  these  forms. 

In  Eczema  marginatum,  however,  the  fungi  are  present 
in  great  number  in  the  scales. 

In  Onychomycosis  trichopJiytina  a  luxuriant  growth  of 
spores  is  seen  besides  the  threads  of  mycelium. 


FIG.  41.— Epidermal  Scales. 


Cultural  Behavior. — Trichophyta  grow,  in  contrast  to 
favus  fungi,  as  well  at  20°  to  24°  C.  as  at  body-tempera- 
ture, and  flourish  upon  media  poor  in  nitrogen,  but  rich 
in  carbohydrates.  Gelatine  is  liquefied.  The  cultures  are 
remarkable  for  their  great  pleomorphism;  the  formation 
of  pigment  varies  greatly  in  colonies  from  one  and  the 
same  stock.  On  agar  trichophyta  form  stars  with  many 
long  rays  radiating  from  a  centre,  which  may  be  of  vary- 
ing appearance.  It  may  be  pyramidal,  concave,  or  con- 
vex. The  surface  of  the  colony  often  appears  as  if  pow- 


SKIN   DISEASES  315 

dered  with  flour,  and  occasionally  a  down  of  air-mycelium 
is  formed.  The  colonies  may  be  yellow,  pink,  violet, 
brownish-red,  or  brownish-black. 

Differential  Diagnosis. — It  is  only  possible  in  a  limited 
number  of  cases  to  differentiate  between  favus  and  tricho- 
phytosis  by  means  of  microscopical  examination  alone. 
If  the  typical  favus  products,  the  scutula,  are  present, 
microscopical  examination  reveals  a  very  characteristic 
picture;  but  in  just  those  cases  in  which  the  clinical  diag- 
nosis lies  between  favus  and  trichophytosis,  as  a  rule,  so 
few  fungi  can  be  detected  that  the  points  characteristic 
of  favus  fungi,  in  contrast  to  trichophyta — namely,  their 
greater  variety  of  form,  their  thicker,  more  gnarly  threads, 
with  numerous  septa,  and  giving  off  branches  more  at  a 
right  than  at  an  acute  angle — are  not  sufficiently  promi- 
nent in  microscopical  specimens  to  allow  of  a  diagnosis. 

Cultural  procedures  are  also  often  of  no  aid  in  these 
cases,  since  undoubted  reproductive  organs,  which  ordi- 
narily make  it  possible  to  distinguish  the  various  types  of 
hyphomycetes,  are  not  known  in  the  fungi  of  the  skin; 
and  the  macroscopical  appearance  of  the  cultures  differs 
so  widely  under  the  influence  of  various  factors,  that  the 
cultures  of  favus  fungi  and  trichophyta  may  resemble  each 
other  very  closely.  Finally,  the  results  of  animal  inocu- 
lation are  of  diagnostic  value  only  when  positive.  In 
many  cases  bacteriological  examination  is  of  diagnostic 
value  only  in  so  far  as  it  may  establish  the  fact  that  a 
dermatomycosis  is  present. 

Pityriasis  Versicolor 

Pityriasis  versicolor  is  excited  by  the  Microsporon 
furfur.  The  micro-organism  is  present  only  in  the  horny 
layer  of  the  skin.  If  the  scales  are  examined  in  potassium 


316    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

hydrate,  glycerine,  or  water,  numerous  fungi  are  seen,  in 
the  form  of  short  U-shaped  threads,  with  few  branches, 
between  which  groups  of  spores  are  visible. 

The  microscopical  picture  is  so  characteristic  that  the 
use  of  cultural  methods  is  not  necessary  for  diagnostic 
purposes. 

Cultivation  of  the  fungi  from  the  scales  is  very  diffi- 
cult. If,  however,  they  have  been  cultivated,  the  follow- 
ing generations  grow  easily  on  the  usual  culture  media, 
both  at  room-  and  at  body-temperature.  Before  collecting 
the  scales  for  cultivation,  the  skin  is  disinfected  with 
bichloride  of  mercury  washed  with  water,  and  sponged 
with  a  mixture  of  alcohol  and  ether.  The  scales  are 
rubbed  according  to  KrdVs  method  and  planted  upon 
urine  agar  (1  to  10)  or  Fingers'  epidermin  agar. 

Erythrasma 

Erythrasma  is  excited,  according  to  the  opinion  of 
most  authors,  by  the  Microsporon  minutissiinum.  This 
micro-organism  also  develops  in  the  horny  layer  of  the 
skin.  The  scales  are  best  stained  according  to  Bizzozercfs 
method,  and  examined  with  the  oil-immersion.  The  fungi 
are  conspicuous  for  their  exceptional  delicacy.  Long, 
winding  threads,  with  many  septa  are  seen,  lying  close 
together.  Among  the  threads  of  mycelium  numerous 
spores  of  varying  shape  are  seen,  which,  because  of  their 
minuteness,  may  be  mistaken  for  cocci. 

Attempts  to  cultivate  the  fungus  have  not  succeeded. 


SKIN  DISEASES  317 

EXAMINATION  FOR  SPIROCHETA  PALLIDA 

The  spirochetae  may  be  demonstrated  in  the  smear  in 
the  cut  section,  and  in  the  fresh  material.  The  animal  ex- 
periment is  not  yet  practical,  nor  have  means  been  found 
to  culture  the  spirochete. 


^^^^^•^•^^^^ 

FIG.  42.-Spirochetae  from  a  Broad  Condyloma,  Magnified  1,500 
ss;  a,  Spirocheta  Pallida,  6,  Spirocheta  Refringens. 


:    Spirochet*  pallid*  have  been  cultivated  to  a  number 

f  generations,  and  animal  experimentation  successfully 

conducted   at  the    Rockefeller    Institute  in  New  York 

Noguchi:  Journal  of  Experimental  Medicine,  Vol.  XIV, 

Jo.  2,    1911,  and  Jour.  Am.  Med.   Ass'n,   July  8,   1911. 

ipgucU  has  obtained  the  spirochete  pallid*  in  pure  cul- 

re,  and  he   considers  it  as  established  that  testicular 

uons  produced  in  rabbits  by  means  of  syphilitic  mate- 


318    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

rials  are  the  result  of  the  multiplication  of  the  pallida 
and  not  of  some  associated  indefinite  parasite. 

Method  for  Obtaining  the  Material  for  Observation 

Chancres  and  eroded  papules  are  first  thoroughly  cleaned 
with  a  piece  of  absorbent  cotton  which  has  been  saturated 
with  a  physiological  saline  solution  in  order  to  remove  the 
superficial  secretions,  which,  as  a  rule,  contain  but  few 
spirocheta3,  but  many  other  kinds  of  micro-organisms; 
then  they  are  dried.  lodoform  and  other  medications 
must  first  be  removed,  which  is  done  by  rubbing  and  ap- 
plications of  the  physiological  saline  solution.  The  eroded 
surface  is  rubbed  with  a  platinum  loop  until  a  slight 
serum  (irritation  serum)  oozes  out,  which  is  used  for  the 
examination.  A  large  admixture  of  blood  is  to  be 
avoided.  The  secretion  for  the  examination  is  easily 
obtained  by  milking  the  papule  or  the  chancre  with  two 
fingers.  Especially  suitable  for  the  examination  is  the 
scraping  of  the  border  of  the  erosion,  which  scraping  has 
been  done  with  a  platinum  spatula  according  to  Hoffmann. 
A  large  number  of  spirocheta3  is  found  in  the  secretions 
which  were  pressed  out  of  the  excised  primary  lesion. 

In  closed  efflorescences  the  horny  layer  is  removed  with 
a  knife,  care  being  taken  to  avoid  any  hemorrhage,  and 
the  secretion  is  obtained  from  the  border  zone. 

In  pustules  and  pemphigus  the  blebs  are  first  opened 
and  the  secretion  obtained  from  the  bottom. 

Hoffmann's  Method  of  Obtaining  Material  for  Examination 
from  Glands 

The  skin  over  the  inguinal  glands  is  first  shaved,  dis- 
infected and  washed  with  a  physiological  saline  solution. 
A  syringe  of  5  cc  is  then  used  which  has  a  long  cannula 
and  whose  piston  has  asbestos  packing.  The  syringe  is 


SKIN   DISEASES  319 

first  cleansed  with  a  sterile  saline  solution.     The  gland  is 
then  held  with  the  left  hand  and  the  cannula  inserted  into 
it.      We  carefully  attempt  to   introduce  the   aspirating 
needle  into  the  substance  (RhulenscUcU)  of  the  largest 
gland  (in  the  direction  of  the  long  diameter)  and  then 
>egm  to  aspirate,   gradually  removing  the  point  of  the 
eedle.    If  we  don't  obtain  enough  of  secretion,  the  needle 
forced  into  a  neighboring  gland  or  several  glands  until 
we  get  a  few  light  red  drops.     We  know  that  the  needle 
i  m  the  gland,  if  we  move  the  gland  and  the  needle  fol- 
lows the  motion.      The  substance  so  obtained  from  the 
gland  is  sqmrted  into  a  small  sterile  dish;  the  single  drop- 
9  are  thoroughly  mixed   together.      If  necessary    the 
ishes  are  covered,  and  then  thin  smears  are  prepared  as 
rapidly  as  possible. 

Blood  for  the  purpose  of  examination  is  obtained  bv 

puncturing  either  the  vein  or  the  ear.     At  least  1  cc  of 

;ood  is  removed  and  mixed  with  ten  times  its  quantity 

>t  J  per  cent,  solution  of  acetic  acid.     The  dissolved  blood 

centnfuged  and  the  sediment  examined. 

The  Preparation  of  Stained  Specimens 

For  the  preparation  of  smears  we  use  either  cover- 
glasses  or  slides  which  have  been  kept  for  several  days  in 
a  solution  of  equal  parts  of  ether  and  alcohol ;  these  are 
len  most  carefully  cleaned.     A  drop  of  the  serum  to  be 
•xamined  w  taken  up  with  the  margin  of  a  cover-glass 
ch  is  then  rapidly  moved  upon  the  slide  from  left  to 
right,  the  cover-glass  being  kept  inclined.     In  this  way 
we  get  a  thin  smear  of  equal  thickness,  which  is  fixed  by 
immersion  in  absolute  alcohol  for  ten  minutes  and  osmic 
acid  vapors.     The  lattermay  be  performed  in  the  fixation 
bes  of  Hamm.     One  end  of  this  tube  is  filled  with  glass 
wool  which  is  saturated  with  a  solution  of  1  per  cent 


320    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

osmic  acid  in  a  solution  of  1  per  cent,  chromic  acid.  The 
slide  is  first  put  into  the  tube  for  one  to  two  minutes,  after 
which  the  smear  is  prepared,  and  the  wet  smear  is  exposed 
to  the  fumes  of  the  osmic  acid  for  twenty  to  forty  seconds  ; 
it  is  then  dried  in  the  air,  after  which  it  is  passed  three 
times  through  the  flame. 

The  fixation  with  the  osmic  acid  fumes  can  be  done 
also  in  this  way:  Into  a  double  dish  is  put  an  open  glass 
dish  of  5  cm  diameter,  into  which  are  put  5  cc  of  a  1  per 
cent,  solution  of  osmic  acid  and  10  drops  of  glacial  acetic 
acid.  The  slide  is  put  into  this  glass  dish  before  the 
smear  is  made  and  exposed  to  the  osmic  acid  fumes  for  two 
minutes.  The  smear  is  then  quickly  made  over  the  sur- 
face which  was  exposed  to  the  osmic  acid,  and  the  slide, 
still  wet,  again  exposed  to  the  fumes  of  the  osmic  acid  for 
another  one  to  two  minutes.  After  the  specimen  has 
dried  in  the  air,  it  is  put  into  a  very  light  red  solution  of 
permanganate  of  potash  for  one  minute,  then  washed 
with  water  and  dried  between  filter-paper. 

Method  of  Staining 

There  are  a  great  many  methods,  but  the  best  is  with 
Giemsa  solution  manufactured  either  by  Gruebler  of 
Leipzig,  or  by  Leitz  of  Berlin. 

Preparation  of  the  Staining  Fluid 

Ten  drops  of  Giemsa  solution  are  shaken  up  in  a  glass 
beaker  with  10  cc  of  distilled  water  which  is  free  from 
acids.  Care  must  be  taken  that  the  staining  pigment  is 
not  precipitated  while  shaking.  This  solution  must  be 
prepared  freshly  before  use. 

The  Staining 

The  specimen  is  put  into  a  flat  glass  dish,  the  smear 
downward  and  the  staining  solution  is  poured  over  it 


SKIN   DISEASES  321 

The  cover-glasses  are  kept  on  glass  rods.  The  duration 
of  staining  is  one  to  two  hours.  Before  the  specimen  is 
removed  from  the  staining  solution,  a  thin  membrane 
which  formed  on  the  surface,  is  first  removed  with  filter- 
paper.  The  specimen  is  then  washed  with  water  and 
dried  between  filter-paper. 

Method  for  Quick  Staining 

The  specimen  is  covered  with  the  diluted  Giemsa  solu- 
tion and  held  over  a  flame  until  it  steams.  After  a  quar- 
ter of  a  minute  the  staining  fluid  is  poured  off.  This  pro- 
cedure is  repeated  four  times,  but  at  the  fourth  time  the 
staining  fluid  remains  on  for  one  full  minute.  Thereupon 
the  specimen  is  washed  with  water  and  dried  with  filter- 
paper.  In  a  well-stained  specimen  with  Giemsa  solution 
the  spirochetse  are  stained  a  distinct  red  and  the  leuco- 
cytes a  very  dark  red ;  in  unsuccessful  staining  the  color- 
ing appears  blue.  Since  the  spirochetae  are  found  mostly 
in  the  neighborhood  of  the  red  blood-cells,  we  look 
under  the  microscope  for  these  first.  For  the  examina- 
Jion  oil  immersion  is  used  and  a  strong  ocular  (com- 
pensation ocular  4). 

Examination  of  the  Fresh  Specimen 

A  small  drop  of  the  material  to  be  examined  is  put  on 
a  cover-glass,  a  little  of  a  physiological  salt  solution  is 
added,  the  cover-glass  is  then  put  on  a  slide  and  the  mar- 
gins of  the  cover-glass  are  smeared  with  vaseline  or  wax. 
Hanging-drop  examinations  can  also  be  made,  but  tke 
drop  must-be  as  flat  as  possible.  Apochromatic  and  com- 
pensation ocular  6  to  12  are  used.  Strong  illumination  is 
necessary.  The  spirocheta3  are  found  more  easily  in  the 
dark  field  illumination. 


322    CHEMISTRY,.  MICROSCOPY,  AND  BACTERIOLOGY 

Examination  of  Cut  Sections 

The  old  method  of  Levaditi  is  the  best. 

The  slices  to  be  imbedded  should  be  no  thicker  than 
2  mm.  All  apparatus  to  be  used  must  be  scrupulously 
clean. 

1.  Fixation  for  twenty- four  hours  in  a  10  per  cent, 
solution  of  formalin  (longer  fixation  does  not  hurt). 

2.  Hardening  for  about  twelve  hours  in  95  per  cent, 
alcohol. 

3.  Washing  repeatedly  in  distilled    water   until  the 
pieces  sink  to  the  bottom. 

4.  Impregnation  with   J  to  8  per  cent,  silver  nitrate 
solution  which  is  contained  in  a  wide-mouthed  100  cc 
flask  and  left  in  the  incubator  at  35°  to  37°  C.  for  three 
to  five  days.     It  is  best  to  renew  the  silver  solution  every 
day. 

5.  Reduction  of  the  pieces  remaining  in  the  flask,  after 
the  silver  solution  has  been  poured  off  with  the  following 
solution : 

Pyrogallol  .  .  .4.0 
Formalin  .  .  .  .  5.0 
Aq.  dest 100 

In  this  solution  the  pieces  remain  for  forty-eight  hours  at 
room-temperature.  This  solution  must  be  prepared  freshly 
every  time  and  renewed  daily. 

6.  Washing  with  distilled  water. 

7.  Hardening  in  alcohol  the  strength  of    which  has 
been  gradually  increased,  and  imbedding  in  paraffin.     The 
slices  must  not  be  any  thicker  than  5  to  10  inicromilli- 
metres.     After-staining  is  not  necessary. 

The  spirocheta3  appear  very  dark,  almost  black,  the 
tissues  are  yellow. 


SKIN   DISEASES  323 

Other  Methods  of  Staining  Spirochetae  Pallidae  (Spirochetes: 
Bosanquet) 

Davidsohn  recommends  the  use  of  cresyl  violet. 
Oppenheimer  and  Sachs  use  carbolic  gentian  violet  (con- 
centrated alcoholic  gentian  violet  solution,  10  cc,  solution 
of  phenol,  5  per  cent. ,  100  cc.  Proca  and  VasiUscu  use 
Gino  de  Rossi's  cilia  stain  (dissolve  fifty  gm.  pure  phenol 
and  forty  gr.  tannin  in  100  cc  water  and  add  tc-  this 
2.5  gm.  basic  fuchsin  dissolved  in  100  cc  absolute  alcohol. 
Stain  in  this  for  ten  minutes,  wash  and  dry.  Then  stain 
with  a  mixture  containing  concentrated  alcoholic  gentian 
violet,  10  cc;  phenol,  five  gm. ;  distilled  water,  100  cc. 

Reitmann  advises  that  films  should  be  first  treated 
with  a  5-per-cent.  solution  of  phosphoric  acid  in  water 
for  five  minutes  and  then  stained  with  carbol-fuchsin, 
warmed. 

Goldliorn  uses  a  complicated  preparation  of  polychrome 
methylene  blue  (methylene  blue,  lithium  carbonate  and 
eosin,  and  McNeal  also  uses  methylene  blue  and  eosin 
(crude  methylene  blue,  twenty  parts;  pure  medicinal 
methylene  blue,  ten  parts ;  eosin  (yellowish) ,  twenty  parts ; 
and  pure  methyl  alcohol,  100  parts.  Stain  on  cover-slip 
for  forty- five  to  sixty  seconds ;  wash  in  dilute  solution  of 
sodium  carbonate,  one  drop  of  1-per-cent.  solution  in 
10  cc  water) . 

A  special  method  of  staining  with  India  ink  is  sug- 
gested by  Burri.  For  this  purpose,  ordinary  India  ink 
is  diluted  with  water  (one  part  in  six,  or  one  in  ten) . 
The  solution  is  sterilized  and  allowed  to  stand  for  two 
weeks,  the  supernatant  fluid  being  then  ready  for  use.  A 
loopful  of  suspension  of  the  organisms  is  mixed  with  a 
drop  of  the  ink-solution,  spread  on  a  slide  and  allowed  to 
dry.  The  spirochetae  are  then  easily  seen  as  colorless 


324    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

spirals  on  a  dark  background.  Some  writers  prefer  a 
stronger  solution  of  the  ink,  i.e.,  one  part  in  two  of  water. 
Mandelbaum  stained  living  spirochetae  pallidae  in  a 
hanging  drop  by  adding  a  loopful  of  Loeffler's  methylene 
blue  solution  along  with  a  loopful  of  decinormal  soda 
solution.  Meirowsky  makes  a  paste  of  methyl  violet  and 
salt  solution,  and  rubs  it  into  the  previously  cleaned  sur- 
face of  the  chancre.  In  the  serum  which  exudes,  there  are 
found  stained  specimens  of  spirocheta  pallida  and  spiro- 
cheta  refringens.  Certain  more  deeply  staining  dots  in 
the  substance  of  the  organisms  he  regards  as  nuclei. 
Crystal  violet  is  as  efficacious  as  methyl  violet. 

Morphology 

In  the  specimen  which  has  been  well  stained  with 
Giemsa  solution,  the  spirochetse  appear  as  very  fine  red- 
stained  spirals,  the  ends  of  which  are  mostly  very  finely 
pointed.  They  show  from  6  to  20  spirals  the  windings 
of  which  are  at  regular  intervals.  Notwithstanding  the 
numerous  winding  some  spirochetse  appear  almost  like  a 
straight  line.  As  a  rule,  the  spirochetaa  are  found  singly, 
often  they  are  found  also  in  twos,  forming  an  acute  angle 
or  the  one  rapidly  running  after  the  other,  or  they  are 
grouped  in  the  form  of  a  "Y";  occasionally  they  are  in 
heaps,  balls  or  twisted  in  the  form  of  tresses.  In  the  same 
specimen  may  be  found  typical  and  atypical  forms,  the 
windings  of  which  are  in  some  very  short,  and  in  some 
the  windings  are  not  noticeable  at  all,  so  that  they  appear 
like  thin  threads.  The  ends  of  some  spirocheta)  are  coiled 
in  the  form  of  a  spiral  and  in  others  they  are  club-shaped. 
These  may  possibly  be  artifices  which  were  produced  in 
the  preparation  of  the  specimen. 

The  characteristics  of  the  living  spirochetse  are:  they 
are  very  fine,  have  little  refractive  power,  their  numerous 


SKIN   DISEASES 


325 


windings  are  regular,  narrow,  deep  and  almost  straight, 
they  do  not  change  their  form  in  motion  or  when  at  rest. 
This  constancy  of  form  gives  to  the  spirochetse  the  peculiar 
appearance  of  having  been  turned  on  a  lathe.  Toward 
the  end  the  windings  are  not  so  high,  the  ends  as  a  rule 
being  pointed.  The  same  specimen  may  show  long  and 

Epithelium  with  migrating  cells. 


Papillary  bodies  with  vesicles. 

FIG.  43.     After  Blaschko. 

short  spirochetae,  whose  motion  is  within  a  short  radius. 
We  observe  rotatory  'motions  on  their  long  axis,  we  see 
them  move  forward  and  backward,  we  see  their  bodies  flex 
upon  themselves  which  may  be  compared  with  the  bending 
and  the  straightening  out  of  an  elastic  tube. 

Differential  Diagnosis 

In  the  differential  diagnosis  we  have  to  consider  the 
spirocheta  refringens,  ballantidis,  buccalis,  Vincenti,  den- 
tium  and  the  sp.  pallidula  pertenuis.  But  the  sp.  pallida 


326    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

may  be  distinguished  from  these  after  a  little  careful 
study.  The  one  exception  makes  the  sp.  pallidula  found 
in  framboesia  tropica  which,  so  far,  could  not  be  differen- 
tiated morphologically  from  the  sp.  pallida. 

In  a  specimen  stained  with  Giemsa  solution  the  sp. 
pallida  is  red,  but  mostly  the  rest  of  the  spirochetae  are  from 
a  violet  to  a  blue  color.  The  other  varieties  are  mostly 
thicker  and  fatter  as  compared  with  their  length,  while  the 
finer  forms  are  shorter  than  the  sp.  pallida.  They  do  not 
end  in  fine  points  as  the  sp.  pallida,  and  their  windings 
are  flatter  and  irregular.  In  the  fresh  specimen  their 
motility  is  much  greater;  they  are  more  refractive  and 
therefore  more  easily  found  than  the  sp.  pallida.  Further- 
more, they  do  not  show  such  constancy  of  form  as  the 
sp.  pallida;  they  show  their  windings  only  while  in 
motion,  and  when  at  rest  they  straighten  out  more  or  less, 
showing  nearly  a  straight  line.  The  sp.  dentium  shows 
very  great  similarity  to  the  sp.  pallida,  as  it  also  stains  red 
with  Giemsa;  is  also  very  fine,  has  regular  windings,  has 
little  refractive  power,  and  does  not  change  its  form  in 
motion.  But  they  are  to  be  differentiated  from  the  sp. 
pallida,  because  of  their  spirals  not  being  so  deep  as  in 
the  sp.  pallida. 

In  order  to  avoid  mistakes  only  such  spirochetae  should 
be  taken  into  consideration  which  correspond  in  all  respects 
with  the  normal  type,  when  we  are  to  make  the  diagnosis 
from  the  morphological  properties  alone. 


CHAPTER  XII 

THE  USUAL  METHODS  OF  BACTERIOLOGICAL 
EXAMINATION,  FORMULAE  OF  STAINS,  AND 
CULTURE  MEDIA 

I.  Examination  in  a  Hanging-Drop 

For  the  examination  in  a  hanging-drop  a  concave  slide 
is  used.  A  layer  of  vaseline  is  smeared  around  the  margin 
of  the  concavity.  A  drop  of  sterile,  physiological  (0.85 
percent.),  sodium-chloride  solution,  or  bouillon,  is  placed 
with  a  sterilized  platinum  wire  in  the  centre  of  a  cover- 
glass,  which  is  held  in  a  Cornet  forceps,  and  a  very  small 
quantity  of  the  material  containing  the  bacteria  is  placed 
in  it  by  means  of  a  sterile  wire.  If  the  material  is  fluid, 
a  drop  of  it  is  placed  directly  upon  the  cover-glass.  The 
drop  should  be  flat  and  round.  The  cover-glass  is  so 
placed  upon  the  slide  that  the  drop  hangs  free  in  the  con- 
cavity, which  is  completely  closed  by  pressing  the  cover- 
glass  firmly  against  the  vaseline. 

In  the  microscopical  examination  the  concave  mirror 
and  the  iris  diaphragm  are  used.  First,  the  low  power 
and  a  very  narrow  diaphragm  are  used,  and  the  margin  of 
the  drop  is  so  placed  that  it  crosses  the  centre  of  the  field 
as  a  bright  line.  The  diaphragm  is  then  somewhat  opened, 
a  drop  of  cedar-oil  placed  upon  the  cover-glass,  without 
shifting  the  specimen,  and  the  low  power  replaced  by  the 
oil-immersion.  The  margin  of  the  drop  is  again  brought 
into  focus.  The  lens  must  be  carefully  lowered,  in  order 
to  avoid  shattering  the  cover-glass. 

327 


328    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 
II.  Examination  in  Stained  Smears 

1.  Preparation  of  the  Specimens 

1.  The  material  is  smeared  upon  cover-glasses  held  in 
Cornet  forceps.     Fluid  material  is  spread,  directly,  in  an 
even,  thin  layer  over  the  entire  surface  of  the  cover-glass 
by  means  of  a  platinum  wire ;  solid  material  after  being 
mixed  with  a  drop  of  sterile  water. 

2.  The  smear  is  dried  in  the  air.      Drying  may  be 
hastened  by  carefully  warming  the  cover-glass  over  the. 
flame,  with  the  smeared  side  up. 

8.  Fixation.     . 

The  cover-glass,  with  the  smeared  side  up,  is  passed 
through  the  flame  three  times.  For  special  purposes — for 
example,  examination  of  blood — the  smears  are  fixed  by 
placing  in  alcohol  (ten  minutes),  or  in  alcohol  and  ether 
aa  (two  to  ten  minutes).  For  SobernlieMs  method  of  fix- 
ation, cf.  p.  54. 

4.  Staining. 

As  much  stain  as  will  remain  upon  it  without  overflow- 
ing is  dropped  from  a  pipette  or  a  dropping-bottle  upon 
the  cover-glass,  which  is  held  in  a  pair  of  Cornet  forceps. 

The  stain  is  allowed  to  act  at  ordinary  temperature, 
or  is  heated  to  the  steaming-point  over  a  small  flame. 
The  length  of  staining  varies  from  a  few  seconds  to  several 
minutes,  depending  upon  the  variety  of  bacteria  and  the 
method  of  staining. 

5.  Wash  with  water. 

6.  Dry  with  filter-paper. 

7.  Mount  in  Canada  balsam. 

Stained  specimens  are  examined  with  the  oil-immer- 
sion, with  wide  diaphragm,  and  the  plane-mirror.  Low- 
power  oculars  are  always  used  in  examining  the  specimens. 


BACTERIOLOGICAL   EXAMINATION  329 

since  with  high-power  oculars  the  objects,  though  larger, 
are  darker  and  less  distinct. 

2.  Staining  Methods  and  Staining  Solutions 

Bacteria  are  stained  with  basic  aniline  dyes.  Those 
most  frequently  used  are  tuchsin,  methylene  blue,  Bis- 
marck brown,  methyl  violet,  dahlia,  and  gentian  violet. 
Most  bacteria,  with  the  exception  of  the  acid-fast,  stain 
with  dilute  watery  solutions.  As  these,  however,  keep 
but  a  limited  time,  stock  solutions  are  made,  which  can 
be  kept  a  long  while,  and  diluted  each  time  for  use.  All 
staining  solutions  must  be  carefully  filtered. 

STOCK  SOLUTIONS 

Saturated  alcoholic  solutions  of  fuchsin,  methylene 
blue,  and  gentian  violet,  are  made  by  placing  sufficient 
dye  in  a  glass-stoppered  bottle  of  alcohol  so  that  a  portion 
remains  undissolved.  The  solution  is  filtered  from  the 
precipitate.  ZieJiVs  or  CzaplewsJcVs  carbol- fuchsin  is  often 
used  as  stock  solution  for  fuchsin,  borax-methylene  blue 
as  stock  solution  for  methylene  blue. 


ZieliVs  Carrol- Fuchsin 
Fuchsin       .       .       .       1.0 
Alcohol        .       .       .10.0 
Acid.  carb.  liquefact.       5.0 
Aqua  dest.  .       .       .   100. 0 


CzaplewsMs  Carbol- Fuchsin 
Fuchsin  .  .  .  1.0 
Acid.  carb.  liquefact.  5.0 
Glycerine  .  .  .50.0 
Aqua  dest.  .  .  .  100.0 


Borax-Methylene  Blue 
Methylene  blue        .       .       .       .       2.0 

Borax 5.0 

Aqua   dest 100.0 

The  saturated  alcoholic  stock  solutions  are  diluted 
before  using  with  distilled  water,  in  a  test-tube,  until 
they  are  just  transparent. 


330    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

The  dilute  solutions  of  carbol-fuchsin  and  borax- 
methylene  blue  are  made  by  diluting  with  ten  times  the 
volume  of  distilled  water. 

GRAM'S  METHOD 

1.  Carbol-gentian   violet,     three    minutes,     without 
heating. 

2.  LugoVs  solution,  one  minute  and  a  half. 

8.  Ten  per  cent,  acetone-alcohol  as  long  as  clouds  of 
stain  are  given  off  from  the  smear. 

4.  Wash  with  water. 

5.  Bismarck  brown,  one  minute,  or  carbol-fuchsin  in 
a  dilution  of  1 :  20  aqua  dest. ,  five  seconds. 

6.  Wash  with  water,  dry,  etc. 

After  (1)  and  (2)  the  stain  is  poured  off  (do  not  wash 
with  water)  and  the  smear  dried  with  filter-paper. 


Carlol-  Gentian  Violet 
Gentian  violet   .       .       1.0 
Alcohol       .       .       .10.0 
Acid.  carb.  liquefact.       5.0 
Aqua  dest.  .       .       .  100.0 

Acetone  Alcohol 
Acetone       .       .       .10.0 
Alcohol  abs.  ad  100.0 


LugoVs  Solution 
Iodine  ....       1.0 
Potassium  iodide     .       2.0 
Aqua  dest.  .       .       .  800.0 

Bismarck  Broivn 
Bismarck  brown       .  .     1.0 
Alcohol       .       .       .10.0 
Aqua  dest.  .       .       .   100.0 


STAINING  OF  TUBERCLE  BACILLI  AND  OTHER  ACID-FAST 
BACILLI 

(a)  Method  of  Ziehl-Neelson 

1.  Carbol-fuchsin    three     minutes,    heating    to    the 
steaming-point. 

2.  Wash  with  water. 


BACTERIOLOGICAL   EXAMINATION  331 

3.  Twenty  per  cent,  nitric  acid,  three  to  five  seconds. 

4.  Wash  with  water. 

5.  Decolorize  with  60  per  cent,  alcohol. 

6.  Wash  with  water. 

7.  Dilute  methylene-blue  solution  one  minute. 

8.  Wash  with  water,  etc. 
Carbol-fuchsin,  cf.  p.  329 

(£)  Czaplew  ski's  Method 

1.  Carbol-fuchsin,  heating  to  the  steaming-point. 

2.  Pour  off  stain,  but  do  not  wash  with  water. 

3.  Dip  in  fluorescin-methylene  blue  six  to  ten  times. 

4.  Dip  in  a  concentrated  alcoholic  solution  of  methy- 
lene  blue  ten  to  twelve  times. 

If  necessary,  repeat  3  and  4. 

5.  Wash  with  water,  etc. 


Concentrated  Alcoholic 
Solution  of  Methylene  Blue 
Methylene  blue  .       .       5.0 
Alcohol        .       .       .   100.0 
Filter  before  using. 


Fluor  escin- Methylene  Blue 
Yellow        fluorescin 

(Gruebler)     .       .       1.0 
Alcohol        .       .       .   100.0 

Allow  to  stand  one 
to  two  days,  decant 
from  precipitate  and 
add  methylene  blue  .  5.0 

Shake;     allow    to    , 
stand  one  day  and  de- 
cant from  precipitate. 

(c)  Method  of  Fraenkel  and  Goblet 

1.  Stain  with  carbol-fuchsin  for  three  minutes,  with 
the  aid  of  heat. 

2.  Simultaneous  decolorization  and   counter-staining 
with  the  following  mixture : 


332    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Saturated  alcoholic  solution  of  methylene  blue        .     50.0 

Sulphuric  acid 25.0 

Aqua  dest 100.0 

(d)  Pappenheim's  Method 

(Differential  stain  between  tubercle  bacilli  and  other 
acid-fast  bacilli:) 

1.  Stain  with  carbol-fuchsin  for  three  minutes,  with 
the  aid  of  heat. 

2.  Dip  three  to  five  times  in  the  corallin  solution, 
without  previous  washing  with  water. 

8.  Wash  with  water,  etc. 

Corallin 

Corallin 1.0 

Saturated  alcoholic  solution  of  methylene  blue       .  100.0 

Glycerine 20.0 

(e)  Baumgarterfs  Method  for  Differentiating  Lepra  Bacilli 

1.  Stain   with    very    dilute    carbol-fuchsin    for   five 
minutes. 

2.  Decolorize  with  a  solution  of  1.0  nitric  acid  in  10.0 
alcohol  for  twenty  seconds. 

3.  Wash  with  water. 

4.  Counter-stain  with  methylene  blue. 

STAINING  OF  DIPHTHERIA  BACILLI 

(a)  Stain  with  carbol-fuchsin  1  in  10  aqua  dest.  for 
one  minute,  without  heating. 

(b)  Stain  with  Loeffler's  alkaline  methylene  blue  for 
two  minutes,  without  heating. 

Loeffler^s  Alkaline  Methylene  Blue 

Concentrated  alcoholic  solution  of  methylene  blue       80.0 
0. 01  per  cent,  watery  solution  of  potassium  hydrate     100. 0 


BACTERIOLOGICAL   EXAMINATION 


333 


(c)  Stain  according  to  Roux  for  two  minutes,  without 
heating : 


1.  Dahlia  violet  .  1.0 
Alcohol  .  .  .  10.0 
Aquadest.  .  .  100.0 


2.  Methyl  green  .  1.0 
Alcohol  .  .  .10.0 
Aqua  dest.  .  .  100.0 


For  use  one  part  of  stain  1  is  mixed  with  two  parts 
of  stain  2.     This  solution  may  be  kept  on  hand. 

(d)  Neisser's  stain. 

1.  Stain  1  for  twenty  to  thirty  seconds. 

2.  Wash  with  distilled  water. 

8.  Stain  2  for  ten  to  fifteen  seconds. 
4.  Wash  with  distilled  water,  etc. 


1.  Methylene  blue  .  1.0 
Alcohol  .  .  20.0 
Acid.  acet.  glacial  50.0 
Aqua  dest.  .  ad  1000.0 


2.  Bismarck  brown         2.0 
Aqua  dest.      .      ad  1000.0 

(Decomposes  easily.) 


New  Method 

1.  Stain  1  fifteen  to  thirty  minutes. 

2.  Washing  with  water. 

3.  Stain  2  fifteen  to  thirty  minutes. 

4.  Washing  with  water. 

Stain  1  consists  of,  2  parts  of  solution  a  and  1  part  of 
solution  b. 


Solution  a. 

Methylene  blue       .  1.0 

Alcohol      .       .       .  20.0 

Aqua  dest.       .       .  1000.0 

Aqua  acet.  glacial  .  50.0 


•    Solution  I. 

Crystal  violet  .       .  1.0 

Alcohol      .       .       .  10.0 

Aquadest.       .       .  300.0 


Stain  2,  Chrysoidin,  1.0;  Aqua  dest.  fervid.  300.0. 


334    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

STAINING  OF  GONOCOCCI 

(a)  Stain  with  very  dilute  methylene-blue  solution  for 
two  minutes,  without  heating. 

(#)  Stain  according  to  Gram,  cf.  p.  380. 
(c)  Double  staining  methods. 

Pappenheim's  Method  (Krystallowicz*  s  Modification). 

Stain  for  one  minute  with  the  following  solution,  with- 
out heating : 

Methyl  green 0.15 

Pyronin 0.25 

Alcohol 2.5 

Glycerine        .       .       .       .       .  20.0 

Aqua  carbolisat.  2  per  cent,  ad  100.0 

May  and  GruenwaWs  Method 

Stain  for  two  minutes.  The  cover-glass  is  placed  in 
the  solution  unfixed,  and  with  the  smeared  side  down. 
For  the  formula  of  the  stain,  cf.  p.  258. 

STAINING  OF  SPORES 
Klein's  Method 

1.  An  agar  culture  which  contains  spores  is  floated  in 
physiological  salt  solution,  the  mixture  treated  with  an 
equal  quantity  of  carbol-fuchsin,  slightly  heated,  and  set 
aside  for  about  half  an  hour. 

2.  Smears  are  made  from  the  mixture,  allowed  to  dry 
in  the  air,  and  fixed  in  the  flame. 

8.  Decolorize  in  1  per  cent,  sulphuric  acid  for  one  to 
two  seconds. 

4.  Wash  with  water. 


BACTERIOLOGICAL   EXAMINATION  335 

5.  Stain  with  dilute  methylene  blue  for  three  to  four 
minutes. 

The  spores  are   stained  red,  the  bacilli  blue. 

STAINING  OF  THE  CAPSULES  OF  ANTHRAX  BACILLI 
(a)  Johne's  Method 

1.  Stain  with  a  2  per  cent,  watery  solution  of  gentian 
violet  for  two  minutes,  heating  carefully. 

2.  Wash  with  water. 

3.  Decolorize  with  1  to  2  per  cent,  acetic  acid  for  six 
-to  ten  seconds. 

4.  Wash  with  water. 

Examine  in  water,  not  in  Canada  balsam. 

(fl)  RaeUger's  Method 

1.  Stain     with     formalin-gentian  violet    for    twenty 
seconds  without  previous  fixation. 

2.  Wash    with    water,   dry,   and    mount  in   Canada 
balsam. 

Formalin-gentian  violet. 

Fifteen  to  twenty  grammes  of  gentian  violet  are  covered 
with  100  to  200  cc  of  formalin,  the  mixture  thoroughly 
tirred,  allowed  to  stand  several  hours,  and  filtered. 

(c)  Hamm's  Method 

1.  Fixation  in  osmium  fumes  (cf.  p.  320). 

2.  Spreading  of  the  bacteria  in  ascitic    fluid      The 
material  is  carefully  rubbed  up  with  a  drop   of  ascitic 

m  spiral  motions.  Staining  ten  to  fifteen  min- 
utes with  a  dilute  Giemsa  solution  (10  drops  to  10  cc 
aqua  dest.),  warming  it  slightly  the  last  three  to  five 
minutes. 


336    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

STAINING  OF  PLAGELLA 

For  the  demonstration  of  flagella  a  very  dilute  mix- 
ture of  bacteria  is  made  from  a  young  agar  culture,  and 
spread  in  a  very  thin  smear  upon  cover-glasses  which  are 
absolutely  clean  and  free  from  fat. 

In  order  to  avoid  overheating,  the  smear  is  held  in  the 
fingers  when  passed  through  the  flame  for  fixation. 

(a)  Loeffler^s  Method 

1.  Fix  the  flagella,  heating  just  to  the  steaming-point. 
(For  the  mordant,  see  below. ) 

2.  Wash  with   water  until  the   mordant  is  entirely 
removed. 

3.  Wash  with  alcohol. 

4.  Stain  with  aniline-water-fuchsin  solution,  to  which 
1  per  cent,  sodium  hydrate  has  been  added  until  precipi- 
tation commences,  one  minute,  heating  to  the  steaming- 
point. 

5.  Wash  with  water,  dry,  mount  in  Canada  balsam. 


Mordant 


Twenty  per   cent,   solution 

of  tannin,  10  cc. 
Cold  saturated  solution  of 

ferrous  sulphate,  5  cc. 
Watery  or  alcoholic  solution 


For  some  bacteria  alkali  (a 
few  drops  of  a  1  per  cent, 
solution  of  NaOH)  must 
be  added  to  the  mordant ; 
for  other  acids  (H2S04). 


of  fuchsin,  1  cc. 

Aniline  Water 

Five  parts  of  aniline  oil  are  added  to  100  parts  of 
water,  the  mixture  shaken  thoroughly,  and  filtered  through 
a  moist  filter.  The  filtrate  must  be  absolutely  clear.  The 
dye  is  either  dissolved  in  the  aniline  water  directly,  or 


BACTERIOLOGICAL   EXAMINATION  337 

sufficient  of  a  concentrated  alcoholic  solution  of  the  dye  is 
added  to  the  aniline  water  to  produce  a  distinct  opal- 
escence. 

(b)  Bunge*s  Method 

1.  Fix  the  flagella  for  one  to  five  minutes  with  the  aid 
of  heat. 

2.  Wash  with  water. 

8.  Dry  between  filter-paper. 

4.  Stain  with  carbol-gentian  violet  with  the  aid  of 
heat. 

5.  Wash  with  water,  etc. 

Mordant.—  Three  parts  of  a  concentrated  watery  solu- 
tion of  tannin  are  mixed  with  1  part  of  a  1  to  20  watery- 
solution  of  liquor  ferri  sesquichlor. ;  1  cc  of  a  concentrated 
watery  solution  of  fuchsin  is  added  to  10  cc  of  this  mix- 
ture. 

The  mordant  must  stand  several  days.  Each  time 
before  using  H202  is  added,  a  drop  at  a  time,  until  the 
solution  is  reddish-brown. 

Other  methods  have  been  suggested  by  Van  Ermengen^ 
Zettnow,  and  others. 

STAINING  OF  FUNGI 

(a)  Bizzozero's  Method,  modified  ~by  Plauth 
"  Scales  are  placed  in  glacial  acetic  acid  on  a  slide  and 
crushed  with  a  second  slide.  Harden  and  dehydrate  with 
alcohol;  heat  until  the  alcohol  and  acetic  acid  have  evap- 
orated and  the  scales,  still  somewhat  moist,  lie  upon  the 
dry  slide.  Stain  with  ZieliTs  solution  for  three  minutes. 
Remove  the  solution  carefully  with  a  piece  of  filter-paper. 
Iodine-potassium  iodide  solution  (1:2:  800)  for  one  min- 
ute. Decolorize  with  aniline  oil  until  no  more  clouds  of 
stain  are  given  off.  Examine  in  aniline  or  xylol.  The 


338     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

fungi  appear  dark  red,  the  tissue  pale  pink."  (Plauth 
in  "Handbuch  d.  pathog.  Mikro-org.  v.  Kolle  und  Was- 
sermann.") 

(b)  WadscWs  Method    . 

1.  Mixture  of  aniline  water  (cf.  p.  836)  and  a  concen- 
trated alcoholic  solution  of  gentian  violet  (2:1)  for  ten 
to  fifteen  minutes. 

2.  Mixture  of  equal  parts  H202  and  5  percent,  watery 
solution  of  potassium  iodide,  three  minutes. 

3.  Decolorize  Completely  with  aniline  oil,  to  which 
1  per  cent.  HC1  has  been  added  (thick  scales,  nails,  and 
hair,  eight  to  ten  hours ;  thin  scales  and  microtome  sec- 
tions, two  to  six  hours) . 

4.  Wash  in  xylol. 

5.  Mount  in  Canada  balsam. 

(Microtome  sections  may  be  previously  stained  with 
picrocarmin. ) 

(c)  Kueline  and  Weigerfs  Method 

1.  Crystal  violet  (cf.  p.  342)  about  five  minutes. 

2.  LugoVs  solution  until  stained  black   (one  to  two 
minutes)* 

3.  Dry  with  filter-paper. 

4.  Aniline  oil  until  no  more  dye  is  given  off. 

5.  Xylol  (to  remove  the  aniline  oil) . 

6.  Canada  balsam. 

STAINING  OF  BLOOD  SPECIMENS 

(a)  Hanson's  Method 

1.  Stain  with  borax-methylene  blue  (cf.  p.  829), 
which  has  been  diluted  until  just  transparent  when  exam- 
ined in  a  test-tube,  five  to  ten  seconds. 

(The  specimen  is  dipped  into  the  staining  solution.) 


BACTERIOLOGICAL   EXAMINATION  339 

2.  Wash  in  a  glass  of  ordinary  water  until  the  speci- 
men shows  a  greenish  tinge. 

3.  Dry,  and  mount  in  cedar-oil. 

(b)  May  and  GruenwalcTs  Method  (cf.  p.  258) 

Stain  two  minutes.  The  cover-glass  is  placed  in  the 
staining  solution  with  the  smeared  surface  down. 

(c)    Giemsa's  Method  (a  Modification   of  RomanowsM  s 
Method) 

Stock  solutions :  1  per  cent,  watery  solution  of  eosin, 
0.08  per  cent,  watery  solution  of  azur  ( Hoechst). 

Preparation  of  staining  solutions:  1  cc  of  the  1  per 
cent,  eosin  solution  is  added  to  200  cc  of  aqua  dest.  To 
9  cc  of  this  solution  1  cc  of  the  0.08  per  cent,  azur  solu- 
tion is  added. 

The  specimen  is  floated  upon  this  mixture  in  a  watch- 
glass.  The  length  of  staining  varies  from  ten  minutes  to 
several  hours.  The  staining  is  controlled  with  the  micro- 
scope by  examining  the  smear  mounted  in  water  (with 
the  dry  system).  On  the  appearance  of  precipitation 
of  the  stain  the  smear  is  washed  with  80  to  40  per  cent, 
alcohol. 

III.  Examination  of  Cut  Sections 

The  pieces  of  tissue  are  hardened  in  alcohol. 
EMBEDDING  IN  PARAFFIN 

1.  Place  in  aniline  oil  until  the  specimen  is  trans- 
parent.    (Place  in  a  closed  glass  in  the  paraffin  oven.) 

2.  Place  in  xylol,  which  is  repeatedly  changed,  until 
the  xylol  no  longer  turns  yellow  (about  one  hour). 

3.  Dry  with  filter-paper. 

4.  Place  in  fluid  paraffin  (melting-point,  56°  C. )  in  a 


340    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

thermostat  at  54°  C.  The  paraffin  is  changed  once.  Stay 
in  the  thermostat  one  to  four  hours,  according  to  the  size 
of  the  specimen. 

5.  The  specimen  and  paraffin  are  placed  in  an  embed- 
ding frame.  The  paraffin  is  quickly  solidified  by  cover- 
ing it  with  water  or  placing  it  in  an  ice-chest. 

The  block  of  paraffin  is  suitably  cut,  fastened  upon  a 
block  of  wood  by  melting  slightly,  and  cut  with  a  micro- 
tome with  a  dry  knife. 

The  individual  sections  are  taken  from  the  knife  and 
placed  directly  upon  a  slide,  which  has  been  smeared  with 
glycerine-albumin,  and  moistened  with  water.  The  water 
is  poured  off,  the  remainder  absorbed  with  filter-paper, 
and  the  slide  placed  in  an  incubator.  After  twelve  hours 
the  sections  are  treated  in  the  following  manner: 

1.  Remove  the  paraffin  by  placing  in  xylol. 

2.  Place  in  absolute  alcohol. 

3.  Place  in  96  per  cent,  alcohol. 

4.  Place  in  water. 

5.  Stain. 

6.  Wash  in  water. 

7.  Dehydrate  in  alcohol. 

8.  Clear  in  xylol. 

9.  Mount  in  Canada  balsam. 

Glycerine- Albumin  Solution 

A  measured  quantity  of  egg-albumin  is  beaten  to  a 
froth,  an  equal  quantity  of  pure  glycerine  added,  and  the 
mixture  filtered. 

EMBEDDING  IN  CELLOIDIN 

Two  solutions  of  celloidin  are  made  in  alcohol  and 
ether  aa,  a  thin  solution  and  a  thick,  syrupy  solution. 


BACTERIOLOGICAL   EXAMINATION  341 

The  specimens,  which  should  not  be  thicker  than  1 
centimetre,  ar  aken  from  the  absolute  alcohol,  and  placed 
for  at  least  twenty- four  hours  in  the  thin  solution  of  cel- 
loidin,  and  then  for  the  same  length  of  time  in  the  thick 
solution.  They  are  then  placed  on  a  cork,  gradually 
covered  with  the  thick  solution,  and,  in  order  to  prevent 
too  rapid  evaporation,  covered  with  a  glass  bell.  When 
the  celloidin  is  sufficiently  dry,  the  specimens  are  placed 
for  twenty-four  hours  in  80  per  cent,  alcohol. 

When  cutting  the  specimens,  the  knife  and  specimens 
are  moistened  with  alcohol. 

FURTHER  TREATMENT  OF  THE  SECTIONS 

1.  Place  in  dilute  alcohol. 

2.  Stain. 

8.   Dehydrate  in  96  per  cent.,  then  in  absolute  alcohol. 

4.  Clear  in  xylol. 

5.  Mount  in  Canada  balsam. 

UNIVERSAL  STAINING  METHODS  FOR  DEMONSTRATING 
BACTERIA  IN  SECTIONS 

Loeffler's  Method 

1.  Stain  in  Loeffler's  methylene  blue  three  to  five  min- 
utes. 

2.  Differentiate  in  0.5  to  1  per  cent,  acetic  acid  ten  to 
twenty  seconds. 

8.   Dehydrate  in  alcohol,  xylol,  Canada  balsam. 

Staining  with  Gentian  Violet 

1.  Stain  in  a  2  per  cent,  watery  or  alcoholic  solution 
of  gentian  violet  until  the  sections  are  dark  violet. 

2.  Wash   in  absolute    alcohol  until  the  sections  are 
light  violet. 

8.  Clear  in  xylol,  and  mount  in  Canada  balsam. 


342    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Pfeiffer's  Method 

1.  Stain  in  carbol-fuchsin  (1:  10)  thirty  minutes. 

2.  Differentiate  in  60  per  cent,  alcohol,  to  which   1 
drop  of  acetic  acid  has  been  added,  until  the  sections  are 
grayish-violet. 

8.   Dehydrate  in  absolute  alcohol,  xylol,  Canada  bal- 
sam. 

SPECIAL  STAINING  METHODS 
Gram's  Method 

1.  Stain  with  aniline  water  gentian  violet  (cf.  p.  336) 
five  to  thirty  minutes. 

2.  LugoTs  solution  (cf.  p.  830)  one  to  two  minutes. 

3.  Differentiate  in  absolute  alcohol  until  the  sections 
are  nearly  colorless. 

4.  Wash  in  water. 

5.  Stain   with  Bismarck  brown   (cf.   p.    380)   one  to 
two  minutes. 

6.  Place  in  60  per  cent.,   then  in  absolute  alcohol, 
xylol,  Canada  balsam. 

Kuehne  and  Weigertfs  Method 

1.  Stain  in  lithium  carmin  two  to  three  minutes. 

2.  Wash  in  3  per  cent.  HC1  alcohol  (70  per  cent.). 
8.   Wash  in  aqua  dest. 

4.  Stain  with  crystal  violet  five  to  ten  minutes. 

5.  Treat  with  LugoVs  solution  until  the  sections  be- 
come black  (about  one  to  two  minutes) . 

6.  Dry  with  filter-paper. 

7.  Treat  with  aniline  oil  until  no  more  of  the  dye  is 
given  off. 

8.  Clear  with  xylol,  and  mount  in  Canada  balsam. 


BACTERIOLOGICAL   EXAMINATION  343 

Lithium  Carmin 

Carmin,  2.5  to  5.0;  saturated  watery  solution  of  lith- 
ium carbonate,  100.0. 

Crystal  Violet 

Stock  solution:  Crystal  violet,  1.0;  alcohol,  10.0. 
Staining  solution :  One  cc  of  stock  solution  is  diluted 
with  10  cc  of  aqua  dest. ,  and  treated  with  1  drop  of  HC1. 

STAINING  OF  TUBERCLE  BACILLI 

(a)  1.   Stain  with  carbol-fuchsin  thirty  minutes  (in 
incubator  at  37°  C.). 

2.  Wash  with  water. 

3.  Decolorize  in 3  percent.  HC1  alcohol  (70  percent.). 

4.  Wash  with  water. 

5.  Counterstain  with  dilute   methylene  blue  two  to 
three  minutes. 

6.  Wash  in  water. 

7«  Alcohol,  xylol,  Canada  balsam. 

(b)  1.  Stain  in  carbol-fuchsin  thirty  minutes. 

2.  Decolorize  in  20  per  cent,  nitric  ten  seconds,  and  60 
per  cent,  alcohol  until  the  sections  are  colorless. 

3.  Wash  in  water. 

4.  Counterstain  with  dilute   methylene   blue  two  to 
three  minutes. 

5.  Wash  with  water. 

6.  Alcohol,  xylol,  Canada  balsam. 

STAINING  OF  DUCREY'S  BACILLI 

(a)  Peterson's  Method  for  Paraffin  Sections 
1.  Stain  in   Unna^s  methylene-blue  solution  twenty- 
four  hours. 


344    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

2.  Aniline  oil  about  three  to  four  hours. 

8.  Aniline  xylol  one  and  a  half  to  three  hours. 

4.  Xylol,  Canada  balsam. 

(b)  Kref  ting's  Method  for  Celloidin  Sections 

1.  Stain  on  the  slide  in  Unna's  methylene  blue  two 
to  five  minutes. 

2.  Dry  with  filter-paper. 

3.  Aniline  xylol  two  to  three  hours. 

4.  Xylol,  Canada  balsam. 

UNNA'S  METHYLENE  BLUE 

Methylene  blue, 

Potass,  carbon aa       1.0 

Aquadest. 100.0 

Alcohol 20.0 

M.  coque  ad  reman.    100.0.      Adde 

Methylene  blue, 

Borax  .       .       .       .       .       .       .     aa       1.0 

In  aqua  dest 100.0 

Soluta  misce. 


IV.  Cultural  Methods 

Preparation  of  Culture  Media 

POTATO 

The  potatoes  are  cleansed  with  a  brush  in  running 
water,  the  eyes  cut  out,  peeled,  sliced,  placed  in  Petri 
dishes,  and  steam-sterilized  for  one  hour  on  three  succes- 
sive days. 

Cylinders  may  be  cut  from  the  peeled  potatoes  with  a 
wide  cork-borer,  and  divided  into  halves  by  an  oblique 


BACTERIOLOGICAL   EXAMINATION  345 

cut.  The  wedges  of  potato  so  obtained  are  placed  with 
the  base  down  in  broad  test-tubes,  which  have  a  constric- 
tion about  1  centimetre  above  the  tip  (Roux's  tubes), 
and  sterilized  in  the  above  manner.  Instead  of  Roux's 
tubes  ordinary  test-tubes  may  also  be  used,  in  whose 
tip  a  little  cotton  is  placed  to  absorb  the  condensation 
water. 

The  potatoes  may  be  rendered  surely  alkaline  by  boil- 
ing ten  minutes  in  soda  solution. 

NUTRIENT  BOUILLON 

1.  Lean  chopped  meat  is  covered  with  twice  its  quan- 
tity of  water. 

2.  One  per  cent,  peptone  and  1  to  2  per  cent,  sodium 
chloride  (calculated  according  to  the  quantity  of  water) 
are  added. 

3.  Boil  in  steam-sterilizer  one  to  two  hours  per  litre  of 
fluid. 

4.  Filter  through  a  moist  folded  filter. 

5.  Neutralize  with  a  saturated  soda  solution  or  25  per 
cent,  sodium  hydrate  until  blue  litmus-paper  is  no  longer 
turned  red,  while  red  is  turned  slightly  blue. 

6.  Boil  in  steam- sterilizer  for  one-half  to  one  hour  per 
litre  of  fluid. 

7.  Filter.     The  filtrate  must  be  absolutely  clear. 

8.  Test  the  reaction.     If  this  must  be  corrected  it  is 
necessary  to  again  boil  and  filter. 

9.  Pour  into  test-tubes  which  are  closed  with  cotton 
plugs  and  have  been  sterilized  by  dry  heat  for  half  an  hour 
at  160°  C. 

10.  Sterilize  in  steam-sterilizer   for  half  an  hour  on 
three  successive  days.     During  the  interval  keep  at  room- 
temperature. 


346    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

NUTRIENT  AGAB 

1  to  8.    As  in  preparing  nutrient  bouillon. 
9.  Add  2  per  cent,  finely  cut  or  pulverized  agar;  to 
dissolve,  boil  for  three  to  five  hours  per  litre  of  fluid. 

10.  Clear  by  adding  the  white  of  an  egg,  which  has 
been 'stirred  in  50  cc  of  water,  to  the  culture  medium, 
which  has  been  cooled  to  50°  C. 

11.  Boil  for  two  hours  per  litre. 

12.  Filter  in  steam-sterilizer  (cover  the  funnel  care- 
fully with  filter-paper) . 

13.  Pour  into  sterile  tubes  in  quantities  of  15  cc  per 
tube  (full  tubes),  which  are  later  used  for  making  plates, 
and  in  quantities  of  5  cc  for  making  slanting  agar  tubes. 

14.  Sterilize  as  in  preparing  nutrient  bouillon. 

For  the  filtration  of  the  agar  the  filter  is  prepared  in 
the  following  way  :l  A  piece  of  absorbent  cotton  is  put  into 
the  angle  of  an  enamel  or  other  funnel  which  will  stand 
heat,  and  over  this  a  closely  woven  piece  of  wire  netting. 
A  similar  piece  of  wire  netting  is  placed  over  the  opening 
of  the  funnel;  over  this  a  thin  sheet  of  absorbent  cotton 
and  another  piece  of  wire  netting  over  the  cotton.  With  a 
filter  so  constructed  the  filtration  can  be  accomplished  in  a 
very  short  time  after  the  sediment  has  well  settled  on  the 
bottom  of  the  hot  kettle,  whilst  the  agar  was  clarifying. 

NUTRIENT  GELATINE 

1  to  4.  As  in  preparing  nutrient  bouillon. 

5.  Add  10  to  15  per  cent,  (in  summer)  gelatine. 

6.  Dissolve  by  slight  heating. 

7.  Neutralize  (cf .  Nutrient  Bouillon,  §  5) . 

1  After  the  description  of  a  laboratory  worker  in  Erbacher's 
Institute  for  Medical  Diagnosis. 


BACTERIOLOGICAL   EXAMINATION  347 

8.  Clear  (cf.  Nutrient  Agar,  §  10). 

9.  Boil  for  three-quarters  of  an  hour. 

10.  Test  the  reaction. 

11.  Filter  in  hot- water  funnel. 

12.  Pour  into  tubes. 

13.  Sterilize  in  steam-sterilizer  for  a  quarter  of  an  hour 

on  three  successive  days. 

After  sterilization  solidify  at  once  by  placing  in 
ice-chest,  then  keep  at  room-temperature. 

In  preparing  the  culture  media  1  per  cent,  of  Liebig's 
extract  of  beef  may  be  used  instead  of  meat. 

The  addition  of  sugar  (2  per  cent. )  glycerine  (4  to  6 
to  8  per  cent. ) ,  and  dyes  to  the  culture  medium  is  never 
made  until  just  before  the  medium  is  poured  into  the 
tubes. 

It  is  frequently  necessary  to  give  the  culture  medium 
a  definite  degree  of  alkalinity.  The  necessary  amount  of 
alkali  is  added  to  the  medium  after  the  latter  has  been 
rendered  neutral  to  litmus.  Thus  for  the  cultivation  of 
cholera  vibriones,  gelatine  and  agar-agar,  after  being  ren- 
dered neutral  to  litmus,  receive  for  each  100  cc  3  cc  of 
a  10  per  cent,  solution  of  crystallized  sodium  carbonate. 

PEPTONE  SOLUTION 
(a)  Preparation  of  the  Stock  Solution 

Peptone  sice 100.0 

Sodium  chloride    .       ...  100.0 

Potassium  nitrate         .       .       .  1.0 

Crystal,   sodium  carbonate       .  2.0 

Aqua  dest 1000.0 

Dissolve  by  heating,  pour  into  flasks  (100  cc  per 
flast),  sterilize. 


348    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

(b)  Preparation  of  Peptone  Solution 

Tubes  are  filled  with  a  dilution  of  1 : 9  of  the  stock 
solution  with  water,  10  cc  per  tube,  and  sterilized. 

MILK 

Fresh  skimmed  milk  which  is  amphoteric  to  litmus- 
paper,  is  poured  into  sterile  test-tubes,  and  steam-sterilized 
for  one  hour  on  the  first  day,  and  half  an  hour  on  the  two 
following  days. 

BREAD 

Dry  bread  is  pulverized,  Bread  placed  in  Erlenmeyer 
flasks,  stirred  to  a  thick  paste,  and  sterilized  for  half  an 
hour  in  the  steam-sterilizer  on  three  successive  days. 

THE  NUTRIENT  MEDIA  OF  CONRADI-DRIGALSKI 

Seven  hundred  and  fifty  gr  of  meat  are  boiled  for  one 
hour  with  900  cc  of  water  and  then  filtered.  To  the  fil- 
trate is  added  water  up  to  900  cc,  10.0  peptone  and  5.0 
common  salt  and  boiled  until  the  peptone  has  dissolved; 
then  are  added  30  gr  agar  and,  after  this  has  dissolved, 
8  to  9  cc  of  a  10  per  cent,  solution  of  water-free  soda  is 
added.  Then  it  is  clarified,  boiled  for  one  hour,  filtered, 
to  it  is  added  a  solution  of  10  gr  of  nutrose  in  100  cc  of 
water  thoroughly  mixed,  filled  in  flasks  of  100  cc  and 
boiled  in  a  steam  kettle  for  twenty  minutes  on  two  suc- 
cessive days. 

When  ready  for  use  the  agar  is  dissolved  and  cooled 
down  to  50°  C.  and  mixed  with  the  following  solutions : 

1.  Thirteen  cc  of  a  litmus  solution  +  1.5  gr  of  sugar 
of  milk. 

2.  One  cc  of  a   0.1  per  cent,  freshly  prepared  solu- 
tion of  crystal  violet  (before  adding,  both  solutions  are 


BACTERIOLOGICAL   EXAMINATION  349 

boiled  for  fifteen  minutes  and  cooled  down  to  50°  C.). 
After  mixing,  the  nutrient  medium  is  poured  onto  plates. 

ENDOS'  FUCHSIN  AGAB 

One  thousand  cc  neutral  3  per  cent,    agar  nutrient 

medium. 

Ten  gr.  chemically  pure  milk  of  sugar. 
Five  cc  0.5  per  cent,   alcoholic   solution  of  fuchsin 

(well  filtered). 
Twenty-five  cc  of  a  10  per  cent,  solution  of  natrium 

sulphite   (freshly   prepared  from  natrium   sulphite- 

which  does  not  yet  show  any  surface  changes) . 
Ten  cc  of  a  10  per  cent,  soda  solution. 

Five  hundred  gr.  of  beef  meat  is  boiled  for  one  hour 
with  1  litre  of  water,  then  are  added  10  gr  peptone,  5  gr 
ordinary  salt  and  8  gr  agar  and  again  boiled,  filtered, 
neutralized  and  made  alkaline  by  the  addition  of  10  cc  of 
the  soda  solution. 

Then  are  added  sugar  of  milk  and  the  fuchsin  solution, 
by  which  the  nutrient  medium  is  stained  red;  then  is 
added  the  solution  of  natrium  sulphite,  which  gradually 
decolorizes  the  nutrient  medium  (the  complete  decolori- 
zation  takes  place  after  the  agar  has  fully  cooled) .  Then 
test-tubes  are  filled  and  sterilized  in  the  steam  kettle. 

Gaethgens  adds  to  ,the  fuchsin  0.33  per  cent,  chemi- 
cally pure  crystalline  caffein,  and  makes  it  alkaline  by 
the  addition  of  15  per  cent,  normal  sodium  hydrate  solu- 
tion under  the  neutralizing  point  of  phenolphthalein. 

LOEFFLER'S  MALACHITE  GREEN  AGAR 

Five  hundred  gr  of  meat  are  boiled  with  2  litres  of 
water  for  one  hour,  filtered,  60  gr  agar  are  added  and 
boiled  until  dissolved,  clarified,  and  again  filtered.  If 


350    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

the  agar  does  not  dissolve  well,  14  cc  of  normal  HC1  are 
added,  which  are  neutralized  with  14  cc  normal  potassium 
hydrate  after  the  agar  has  dissolved.  Then  natrium  car- 
bonate is  added  until  it  reacts  neutral  to  litmus.  After 
neutralization  25  cc  normal  soda  is  added  and  the  weak 
alkaline  solution  is  boiled  up.  To  this  boiling  hot  mass 
are  added  200  cc  of  a  10  per  cent,  watery  solution  of  nu- 
trose.  After  it  has  been  boiled  up  again  the  hot  solution 
is  filled  in  flasks  of  100  cc  and  sterilized  for  twenty  min- 
utes on  two  successive  days.  To  100  cc  of  the  liquefied, 
clear  bouillon  nutrose  agar  which  has  been  cooled  down 
to  50°  C.  are  added  before  use  1.5  cc  of  a  0.2  per  cent, 
solution  of  malachite  green  crystals,  chemically  pure. 
The  green  agar  is  poured  into  Petri  dishes  which  are  left 
open  until  it  cools  and  solidifies.  If  cultures  are  intended 
from  faeces,  Loeffler  recommends  the  addition  of  3  per 
cent,  beef  gall.  But  then  1.9  cc  malachite  green  must  be 
added  instead  of  0.5  cc. 

GREEN  SOLUTION  I 

Nutrose,  1.0;  peptone,  2.0;  grape-sugar,  1.0;  sugar 
of  milk,  5.0;  aqua  dest.,  100;  0.2  per  cent,  solution 
malachite  green  crystals,  chemically  pure,  1.0;  normal 
potassium  hydrate,  1.5. 

GREEN  SOLUTION  II 

Nutrose,  1.0;  peptone,  2.0;  sugar  of  milk,  5.0;  normal 
potassium  hydrate,  1.5;  "malachite  green  120,"  2  per 
cent.  8  cc;  aqua  dest.,  100.0. 

The  solutions  were  prepared  from  10  to  20  per  cent, 
stock  solutions  of  the  several  ingredients,  so  that  at  first 
the  peptone,  the  grape-  and  milk-sugar  were  mixed  to- 
gether, then  were  added  the  potassium  hydrate  and  after 
this  the  nutrose,  and  finally  the  green  stain. 


BACTERIOLOGICAL   EXAMINATION  351 

V.  Lingelsheim's  Litmus  Ascites-Sugar  Agar 

Ten  cc  of  a  10  per  cent,  solution  of  the  sugar  to  be 

examined  in   Kubel-Tiemann's  litmus  solution  are  put 

into  test-tubes  and  heated  for  two  minutes  in  a  water-bath 

C.     After  cooling  are  added  to  each  10  cc,  0  5  cc 

normal  soda  solution.     Of  this  1.5  cc  are  added  to  each 

13.5  cc  of  liquid  ascitesagar  (1  partascites,  8  parts  agar). 

e  nutrient  medium  is  poured  into  Petri  dishes. 

NEUTRAL-RED  AGAR 

For  every  100  cc  of  agar,  0.3  gramme  of  grape-sugar 
and  1  cc  of  a  saturated  watery  solution  of  neutral-red  are 
added,  before  the  agar  is  poured  into  tubes. 

PETRUSCHKY'S  LITMUS- WHEY 

Warm  milk  is  diluted  with  an  equal  quantity  of  water 
and  treated  with  sufficient  dilute  HC1  to  precipitate  all  the 
casein.     A  measured  quantity  is  at  first  tested  to  ascer- 
tain how  much  HC1  is  necessary  to  just  coagulate  the  milk 
and  the  amount  necessary  to  coagulate  the  entire  quantity 
is  calculated  from  it.     The  casein  is  removed  by  filtration  • 
the  filtrate  neutralized  with  soda  solution,  boiled  for  one 
to  two  hours  in  the  steam-sterilizer,  and  filtered.     The 
reaction  is  again  tested,  it  is  rendered  exactly  neutral 
and  again  boiled.     It  -is  then  treated  with  a  sterile  tinct- 
ure of  litmus  until  it  is  violet  in  color,  poured  into  tubes 
and  sterilized. 

BARSIEKOW'S  CULTURE  MEDIUM 

Nutrose 10 

Milk-sugar        ....  10 

Sodium  chloride      .  05 

Aqua  dest.         .  '  1QO.O 

Adde  litmus  solution     .  5.0 


352    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Or,  instead  of  milk-sugar  1.0,  grape-sugar  1.0,  or  grape- 
sugar  and  milk-sugar  aa  1.0. 

The  sugar-nutrose  sodium  chloride  solution  is  boiled 
twenty  minutes  in  the  steam-sterilizer,  filtered,  and  after 
the  addition  of  the  litmus  solution  poured  into  tubes  and 
sterilized  twenty  minutes. 

BLOOD-AGAR 

According  to  Pfeiffer  blood-agar  is  prepared  with 
human  or  pigeon  blood.  The  former  is  obtained  by  prick- 
ing the  ball  of  the  finger  or  lobe  of  the  ear,  after  disinfect- 
ing the  skin  with  alcohol  and  ether,  the  latter  from  the 
large  vein  of  the  pigeon's  wing,  which  is  opened  after  the 
feathers  have  been  removed  and  the  skin  cleansed.  The 
drop  of  blood  is  taken  with  the  platinum  loop,  as  it  issues, 
and  smeared  upon  the  surface  of  congealed  agar.  The 
culture  medium  is  placed  for  twenty- four  hours  in  the  in- 
cubator at  87°  C.,  in  order  to  test  its  sterility. 

CZAPLEWSKI'S  BLOOD-AGAR 

Pigeon-blood,  which  has  been  obtained  under  aseptic 
conditions,  is  mixed  in  an  Erlenmeyer  flask  with  lique- 
fied agar,  which  has  been  cooled  to  50°  C.,  thoroughly 
shaken,  and  liquefied  agar  added  until  the  medium  appears 
but  slightly  red.  After  any  clots  which  may  have  formed 
have  been  removed  with  the  platinum  needle,  the  medium 
is  at  once  poured  into  small  Petri  dishes,  or  tubes,  in 
which  it  is  allowed  to  solidify  obliquely.  Before  using, 
the  plates  are  dried,  inverted  and  open,  for  a  short  time 
in  the  thermostat  at  50°  C. 

HESSE'S  AGAR 

Five  grammes  of  sodium  chloride,  10  grammes  of  agar- 
agar,  80  cc  of  glycerine,  and  5  cc  of  normal  sodium  car- 


BACTERIOLOGICAL   EXAMINATION  353 

bonate  solution  are  covered  with  1,000  cc  of  water  and 
boiled  for  two  hours  in  the  steam-sterilizer.  Five  grammes 
of  Heyderfs  food,  mixed  with  water,  are  added,  and  the 
mixture  boiled  for  a  quarter  of  an  hour  in  a  water-bath, 
filtered,  poured  into  tubes,  and  sterilized  in  the  usual 
manner. 

BLOOD  SERUM 

When  possible  the  blood  is  obtained    under  aseptic 
precautions  by  allowing  it  to  run  through  a  sterile  rubber 
tube  and  a  cannula,  which  is  introduced  into  the  carotid 
of  an  animal,  into  sterile  glass  receptacles,  which  can  be 
tightly  closed.     The  receptacles  containing  the  blood  are 
placed  at  once  in  an  ice-chest  (temperature  7°  to  8°  C.). 
After  the  blood  has  coagulated,  the  clot  is  loosened  from 
the  sides  of  the  glass  with  a  sterile  glass  rod.     After  one 
to  three  days  the  serum,  which  has  separated,  is  removed 
with  a  sterile  pipette  and  placed  in  Petri  dishes  (about 
20  cc  per  dish)  and  tubes  (about  5  cc  per  tube).     Serum 
which  is  not  to  be  used  at  once  may  be  placed  in  sterile 
Erlenmeyer  flasks,  and  after  the  addition  of  about  2  per 
cent,  chloroform,  kept  in  an  ice-chest.     The  Petri  dishes 
and  the  tubes  are  placed  for  two  hours  in  a  thermostat  at 
60°  to  65°  C.,  in  order  to  solidify  the  serum.     The  solid- 
ified  serum  is  transparent  and    amber-yellow  in  color. 
The  medium  is  tested  for  its  sterility  by  placing  in  the 
incubator  at  37°  C. 

When  it  is  impossible  to  obtain  the  blood  in  an  asep- 
tic manner,  it  is  obtained  when  an  animal  is  slaughtered, 
from  a  stab-wound  made  through  a  clean,  or  at  least 
moistened,  area,  and  collected  in  sterile  glass  receptacles. 
The  first  blood  which  issues  is  allowed  to  escape,  since 
the  hair,  etc.,  from  the  area  surrounding  the  wound  is 
washed  by  it.  The  further  treatment  oi  the  blood  is  the 


354     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

same  as  that  described  above.  The  serum  must,  however, 
either  be  sterilized  in  its  fluid  state,  by  placing  it  in  a 
thermostat  at  55°  to  58°  C.  for  four  to  five  hours  on  eight 
successive  days,  or  must  be  sterilized,  after  it  has  so- 
lidified, for  two  hours  on  three  successive  days  at  65°  to 
68°  C.  In  order  to  prevent  too  great  drying  of  the  serum, 
a  vessel  of  water  is  placed  in  the  thermostat  with  it.  The 
majority  of  plates  and  tubes  so  prepared  will  be  found 
sterile  when  tested  in  the  incubator. 

LOEFFLER'S  SERUM 

Three  parts  of  ox  or  sheep  serum  are  mixed  with  1  per 
cent,  slightly  alkaline  grape-sugar  bouillon.  The  mixture 
is  coagulated  and  sterilized  in  a  steam-sterilizer,  which, 
in  order  to  avoid  the  formation  of  bubbles  in  the  medium, 
is  so  gradually  heated  that  the  serum  has  solidified  before 
the  water  begins  to  boil.  Neister  has  suggested  a  special 
serum  oven.  The  medium  so  obtained  is  not  always  sterile, 
and  must  therefore  be  tested  before  use  by  placing  in  the 
incubator  at  87°  C.  Serum  tubes  are  sterilized  in  the 
steam-sterilizer  for  a  quarter  of  an  hour  on  the  two  follow- 
ing days.  Plates  must  be  kept  inverted,  since  consider- 
able condensation-water  is  expressed. 

BLOOD-SERUM  AGAR 

Fluid  blood-serum,  which  has  either  been  obtained  in 
an  aseptic  manner,  or  sterilized  by  fractional  sterilization 
at  55°  C.  (eight  days,  four  hours  a  day) ,  is  heated  to  40° 
to  50°  C.,  and  mixed  in  the  ratio  1:  2,  with  2  to  8  per 
cent,  agar  or  glycerine-agar,  which  has  been  melted  and 
cooled  to  50°  C.  The  mixture  is  poured  into  Petri  dishes, 
or  into  obliquely  placed  tubes,  and  allowed  to  solidify, 


BACTERIOLOGICAL  EXAMINATION  355 

WERTHEIM'S  HUMAN  BLOOD-SEBUM  AGAR 

Human  blood  is  obtained  by  venesection  or  from  the 
placenta.  After  the  cord  has  been  tied  or  cut,  the  mater- 
nal end  is  disinfected  with  corrosive  sublimate,  washed 
with  distilled  water,  and  again  cut  above  the  knot.  The 
blood  which  issues  is  collected  in  sterile  flasks,  and  the 
serum  kept  in  its  liquid  state,  with  the  addition  of  chloro- 
form, and  used  for  the  preparation  of  culture  media,  is 
treated  in  the  above  manner  with  agar,  in  the  ratio  of 
1 :  2  or  1 :  3,  shortly  before  it  is  to  be  used.  The  mixture 
is  allowed  to  solidify  in  slanting  tubes. 

ASCITES-AGAR 

The  serous  fluid  obtained  by  puncture  is  treated  with 
2  to  3  percent,  chloroform,  kept  in  a  cool  and  dark  place, 
and  frequently  shaken.  When  the  fluid  has  become  ab- 
solutely clear,  it  is  withdrawn  with  a  sterile  pipette,  and 
placed  in  tubes.  Before  using,  the  chloroform  is  driven 
off  by  heating  to  35°  C.  (in  a  water-bath  or  incubator). 
The  fluid  is  mixed  with  the  agar  in  the  same  manner  as 
in  preparing  blood-serum  agar. 

KIEFER'S  ASCITES-AGAR 

Ascites  fluid  is  heated  to  50°  C.  shortly  before  it  is  to 
be  used,  mixed  with  an  equal  quantity  of  neutral  liquefied 
glycerine-agar,  which  has  been  colored  to  50°  C. ,  and  con- 
tains 3.5  per  cent,  agar,  5  per  cent,  peptone,  0.5  per 
cent,  sodium  chloride,  and  2  per  cent,  glycerine,  and 
poured  into  Petri  dishes.  If  the  ascites  fluid  is  strongly 
alkaline,  the  agar  is  either  not  previously  neutralized,  or 
is  sufficiently  acidified  to  give  the  mixture  a  slight  alka- 
line reaction. 


356     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

BEER- WORT  CULTURE  MEDIA 

After  sterilization  in  a  steam-sterilizer,  the  beer- wort 
is  set  aside  for  some  time,  the  clear  fluid  decanted  into 
tubes  and  again  sterilized.  By  the  addition  of  10  per 
cent,  gelatine,  or  2  per  cent,  agar,  beer-wort  gelatine  or 
agar  are  obtained.  The  culture  media  are  not  neutralized. 

All  culture  media  must  be  tested  as  to  their  sterility 
before  using.  To  this  end  they  are  placed  for  twenty- four 
hours  in  the  incubator.  Culture  media  whose  .base  is  gela- 
tine must  only  be  exposed  to  temperatures  between  20° 
and  25°  C.  Agar-agar  mixtures,  blood-serum,  potato,  and 
fluid  media  may  be  kept  at  higher  temperatures. 

The  Cultural  Methods  Most  Frequently  Employed 

Aerobic  Cultures 

PLATE  CULTURES 

(a)  GELATINE  PLATE  CULTURES. — Three  tubes  of  gela- 
tine are  liquefied  in  a  water-bath  at  80°  to  35°  C.  One  of 
them  is  removed  and  held  by  the  tip  between  the  thumb 
and  forefinger  of  the  left  hand  (with  the  volar  side  up) 
as  obliquely  as  possible;  the  cotton  plug  removed  and 
held  between  the  third  and  fourth  fingers  of  the  left  hand, 
so  that  the  portion  which  belongs  within  the  tube  does  not 
touch  the  skin.  The  material  to  be  inoculated  is  then  in- 
troduced into  the  gelatine  by  means  of  the  platinum  loop, 
which  is  held  like  a  pen,  and  has  been  sterilized  in  the 
flame  and  again  cooled.  Fluid  material  is  mixed  directly 
with  the  gelatine ;  solid  material  is  first  smeared  upon  the 
side  of  the  tube  and  gradually  mixed  with  the  gelatine. 
'After  the  cotton  plug  has  been  singed,  it  is  replaced,  and, 
by  carefully  tipping  and  turning  the  tube,  the  material  is 


BACTERIOLOGICAL   EXAMINATION  357 

distributed  as  evenly  as  possible  throughout  the  fluid 
medium,  without  allowing  the  latter  to  touch  the  cotton 
plug.  The  tube  is  again  held  in  the  above-described  man- 
ner, with  a  second  tube  parallel  to  it.  Both  are  opened, 
and  one  or  more  loops,  depending  upon  the  number  of 
bacteria  contained  in  the  material  to  be  examined,  are 
transferred  from  the  first  to  the  second  tube ;  both  tubes  are 
again  closed,  and  the  first  tube  is  replaced  in  the  water- 
bath.  After  the  contents  of  the  second  tube  have  been 
carefully  mixed,  several  loops  are  transferred  from  it  to  a 
third  tube.  After  the  mouths  of  the  tubes  have  been 
burned  and  allowed  to  cool,  the  inoculated  gelatine  is  then 
poured  into  sterile  Petri  dishes,  whose  covers  are  raised  at 
one  side  only  just  high  enough  to  allow  of  it.  The  gelatine 
is  again  mixed  by  carefully  rocking  the  plates.  The 
plates  are  marked  O  (original  plate)  1  and  2  (first  and 
second  dilution) ,  and  with  the  date  of  inoculation,  allowed 
to  solidify  upon  ice,  and  placed  in  a  thermostat  at  22°  C. 
(b)  AGAR  PLATE  CULTURES. — Agar  may  be  inoculated 
in  the  same  manner  as  gelatine,  but  must  first  be  melted 
in  boiling  water,  and  again  cooled  to  50°  C. 

SURFACE  CULTURES 

The  material  .to  be  examined  is  placed  upon  the  medium 
contained  in  Petri  dishes,  and  spread  evenly  in  all  direc- 
tions over  its  surfac'e  by  means  of  a  platinum  needle, 
which  has  been  bent  so  that  it  is  parallel  to  the  surface, 
or  a  right-angled  glass  spatula.  The  glass  spatula  may 
be  sterilized  by  burning  alcohol  on  it.  If  the  material  to 
be  inoculated  contains  a  large  number  of  bacteria,  it  is 
necessary,  in  order  to  obtain  isolated  colonies,  either  to 
first  mix  it  with  a  sterile  fluid  (physiological  salt  solution 
or  bouillon),  and  use  a  loop  of  the  mixture  for  inocula- 
ting, or  to  smear  several  (three  to  four)  plates,  one  after 


358    CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

another,  with  the  same  loop,  without  touching  it  again  to 
the  material.  The  plates  are  placed,  inverted  and  open,  in 
the  incubator  some  time  before  they  are  inoculated,  in 
order  to  allow  the  condensation- water  to  evaporate.  Petri 
dishes  are  also  inverted  before  inoculating;  the  dish  is 
removed  from  the  cover,  and  the  culture  medium  smeared 
with  its  surface  down. 

INOCULATION  OF  TUBES  CONTAINING  SLANTING  MEDIA 
(AGAR,  BLOOD-SERUM,  ETC.) 

A  small  quantity  of  the  material  to  be  examined  is 
smeared  over  the  surface  of  the  medium  with  a  platinum 
wire,  while  the  tubes  are  held  horizontally.  To  obtain 
isolated  colonies  several  tubes  are  smeared,  one  after 
the  other,  with  the  same  loop. 

STAB  CULTURES 

Tubes  containing  culture  medium,  which  solidified 
while  they  were  in  a  vertical  position,  are  held  horizon- 
tally, and  the  medium  stabbed  with  a  platinum  needle 
carrying  the  bacteria  to  be  cultivated. 

' '  SCHUETTEL "    CULTURES 

The  medium  is  melted  in  a  water-bath  (agar  must  be 
cooled  to  50°  C. ) ,  a  loop  from  a  pure  culture  is  intro- 
duced, the  tube  thoroughly  shaken,  and  the  medium 
allowed  to  solidify  while  the  tube  is  in  a  vertical  position. 

INOCULATION  OF  FLUID  CULTURE  MEDIA 

This  is  accomplished  in  the  same  manner  as  that  of 
melted  gelatine. 


BACTERIOLOGICAL   EXAMINATION  359 

Anaerobic  Cultures 

It  is  advisable  to  add  reducing  substances  such  as  1  to 
2  per  cent,  grape-sugar,  0.3  to  0.5  per  cent,  sodium  for- 
mate, or  0. 1  per  cent,  sodium  indigo-sulphate,  to  culture 
media  which  are  to  be  used  for  cultivating  anaerobic  bac- 
teria. 

Various  methods  are  used  for  cultivating  bacteria  in 
the  absence  of  air. 

(a)  Mechanical  Exclusion  of  Air 

1.  A  thin  sheet  of  mica,  which  must  be  at  least  large 
enough   to   cover   one-third   of   the  surface   of   the  me- 
dium,   is  placed  in  the  centre  of  the   inoculated  agar, 
or  gelatine,   plates,  just  as  the  medium  commences   to 
solidify. 

2.  Inoculation  of  a  Deep  Layer. — Well-filled  agar,  or 
gelatine,  tubes  are  boiled  half  an  hour  in  a  water-bath,  in 
order  to  expel  the  air,  quickly  cooled,  and  inoculated  with 
the  material  to  be  examined.     After  the  medium   has 
solidified  (on  ice) ,  it  is  covered  with  a  layer  of  agar  or 
gelatine. 

For  examination,  the  tubes  are  broken  and  the  medium 
sliced  with  a  sterile  knife. 

To  obtain  pure  cultures,  stab  cultures  in  well-filled 
agar  or  gelatine  tubes,  which  have  been  boiled  and  quickly 
cooled  on  ice,  are  made,  and  the  medium  likewise  covered 
with  a  layer  of  sterile  medium  after  the  inoculation.  The 
inoculation  must  be  made  with  a  long  needle  which 
reaches  deep  down  into  the  medium. 

Material  is  taken  for  examination  from  stab  cultures 
from  the  depths  of  the  stab  canal,  which  is  entered  froni 
above,  without  breaking  the  tubes., 


360   CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

(b}  Removal  of  the  Air  by  Means  of  an  Exhaust  Pump 

Large  tubes  are  drawn  out  into  a  thin  tube  at  a  point 
in  their  upper  third,  filled  with  the  culture  medium  (the 
constricted  point  must  remain  dry) ,  sterilized,  and  inocu- 
lated in  the  usual  manner.  The  cotton  plug  is  then 
pressed  deep  down  into  the  neck  of  the  tube,  and  the  tube 
closed  with  a  tightly  fitting  rubber  stopper,  through  which 
a  right-angled  glass  tube  passes.  The  tube  is  connected 
with  an  exhaust  pump.  During  the  exhaustion  of  the  air, 
the  culture  medium  is  placed  in  a  water-bath  at  80°  to  35° 
C.  for  gelatine,  at  42°  C.  for  agar.  As  soon  as  the  air  is 
exhausted  (after  about  fifteen  minutes),  the  constricted 
part  of  the  tube  is  closed  by  melting. 


(c)  Removal  of  the  Oxygen  from  the  Air  by  Chemical 
Means 

The  inoculated  tube  is  closed  with  a  cotton  plug,  and 
placed  on  a  wire  shelf  in  a  second  wide  tube,  which  con- 
tains an  alkaline  solution  of  pyrogallol.  (For  every  100 
cc  of  air  space  1  gramme  of  pyrogallic  acid,  and  shortly 
before  the  vessel  is  closed,  10  cc  of  a  solution  of  one  part 
liquor  potassse  in  ten  parts  of  water.  The  outer  vessel  is 
hermetically  closed  with  a  rubber  stopper  and  sealed  with 
liquefied  paraffin.  The  absorption  of  the  oxygen  requires 
about  twenty- four  hours,  during  which  time  the  cultures 
are  kept  at  ordinary  temperature. 

This  procedure  may  also  be  "used  for  plate  cultures  by 
using  a  jar  with  a  ground  top. 

(d)  Replacement  of  the  Air  by  Hydrogen 

The  tube  or  flask  containing  the  inoculated  medium  is 
closed  with  a  rubber  stopper,  through  which  pass  two  right- 


BACTERIOLOGICAL   EXAMINATION  361 

angled  glass  tubes,  one  of  which  extends  into  the  culture 
medium,  while  the  other  extends  but  a  trifle  below  the 
stopper.  The  outer  arms  of  the  tubes  are  drawn  out  to 
capillary  tubes.  The  longer  tube  is  joined  to  a  Kipp's 
hydrogen  generator,  and  hydrogen  run  through  it  until 
the  oxygen  is  driven  off.  The  capillary  tubes  are  then 
closed  by  melting. 

For  plate  cultures,  either  Kitasato's  plates  or  BotTcirfs 
apparatus  is  used.  The  latter  consists  of  a  deep  glass 
dish,  containing  a  glass  bell,  which  rests  on  a  metal  cross. 
Two  U-shaped  tubes  pass  at  opposite  sides  of  the  bell 
between  its  rim  and  the  bottom  of  the  dish.  They  serve 
for  the  introduction  of  hydrogen  and  the  exit  of  the  air, 
and  after  the  latter  has  been  completely  driven  off,  they 
are  closed  by  melting.  The  dish  is  filled  with  liquefied 
paraffin,  in  order  to  exclude  air.  The  inoculated  plates 
are  placed  open  upon  a  wire  shelf  within  the  bell.  A 
dish,  containing  an  alkaline  solution  of  pyrogallol,  is 
also  placed  within  the  bell. 


V.  Determination  of  the  Biological  Characteristics 
of  Bacteria 

Detection  of  Peptonizing  Ferments. — This  is  accomplished 
by  means  of  gelatine  stab  cultures ;  gelatine  is  liquefied  by 
the  action  of  peptonizing  ferments. 

Determination  of  Fermentative  Power. — This  is  accom- 
plished by  means  of  stab  cultures  in  sugar-agar  or  by  the 
inoculation  of  sugar-bouillon,  which  is  poured  into  fer- 
mentation flasks.  The  fermentation  flasks,  which  have 
been  sterilized  in  a  dry  sterilizer,  are  filled  with  sterile,  2 
per  cent,  grape-sugar  bouillon,  and  before  using  are  again 
sterilized  for  half  an  hour  in  the  steam-sterilizer. 


362     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

Detection  of  Acid  or  Alkali  Formation. — This  is  accom- 
plished by  the  addition  of  an  indicator — for  example, 
tincture  of  litmus — to  neutral  culture  media.  Petruscli Icy ' s 
litmus  whey  and  Barsiekoitfs  culture  media  are  largely 
used  for  this  purpose. 

Detection  of  Indol  Formation. — Cf.  p.  112. 

Detection  of  Hydrogen  Sulphide  Formation. — A  piece  of 
moistened  lead  paper  is  placed  between  the  cotton  plug  and 
the  culture  tube,  so  that  it  protrudes  into  the  latter.  If 
hydrogen  sulphide  is  formed,  the  paper  turns  black. 

Determination  of  Reducing  Power.— A  dye  which  is 
decolorized  by  reduction  (methyl ene  blue,  litmus,  neutral 
red)  is  added  to  the  sterile  culture  media. 

To  Determine  whether  Bacteria  are  Aerobic  or  Anaerobic 
Inoculation  of  a  Deep  Layer  is  Used. —  (Cf.  p.  859.) 

Determination  of  Toxin  Formation. — Detection  of  extra- 
cellular toxins:  The  culture  fluid,  which  contains  the 
toxins  in  solution,  is  filtered  free  from  bacteria, l  and  in- 
jected into  test-animals  in  measured  doses.  Detection  of 
intracellular  toxins:  The  bacteria  are  cultivated  upon 
solid  media  and  killed,  removed  with  a  normal  loop 
(capacity  of  2  milligrammes)  without  admixture  of 
media,  and  mixed  with  a  measured  quantity  of  sterile 
fluid.  Definite  quantities  of  this  mixture,  or  a  dilution 
of  it,  are  then  used  for  animal  inoculation.  In  this  man- 
ner an  entire  loop,  or  T^,  -j-oVo  >  etc. ,  of  a  loop,  may  be 
injected.  The  bacteria  are  killed  by  two  hours'  stay  in 
the  thermostat  at  60°  C. ,  or  by  chloroform  vapor.  The 
bottom  of  the  cotton  plug  of  the  culture  tubes  is  moistened 

bacteria-proof  filters  (Chamberland  filter,  filters  of  infusorial 
earth,  etc. )  are  used,  through  which  the  fluid  is  drawn  by  means 
of  a  suction-pump. 


BACTERIOLOGICAL   EXAMINATION  363 

with  chloroform,  replaced,  the  tubes  closed  with  double 
rubber  caps,  and  kept  for  several  hours  in  the  incubator 
at  37°  C.  Before  the  test  is  made,  the  fact  that  the  bac- 
teria have  been  killed  must  be  established  by  transplanta- 
tion upon  other  culture  media,  which  must  remain  sterile. 

VI.  Methods  of  Animal  Inoculation 

1.  Cutaneous  Inoculation. 

The  skin  is  shaved,  disinfected,  freed  from  the  disin- 
fectant, and  may  or  may  not  be  slightly  scarified.  The 
material  to  be  examined  is  then  rubbed  into  it  with  a 
sterile  instrument. 

2.  Subcutaneous  Inoculation. 

(a)  Inoculation  by  Injection. — Fluid  material  is  in- 
jected directly  with  a  hypodermic  syringe,  solid  material 
after  admixture  with  sterile  physiological  salt  solution  or 
bouillon. 

(#)  Inoculation  in  a  Pocket  under  the  Skin. — The  skin 
is  disinfected,  raised  with  a  thumb-forceps,  the  fold  thus 
produced  snipped  with  scissors,  and  the  material  to  be 
examined  introduced  through  the  nick.  If  necessary,  the 
wound  is  closed  with  collodion.  Mice  and  rats  are  usually 
inoculated  just  above  the  base  of  the  tail,  guinea-pigs  on 
the  side  of  the  abdomen  or  chest,  rabbits  on  the  inner  side 
of  the  ear. 

8.  Inoculation  in  the  Large  Cavities  of  the  Body. 

A  slight  nick  is  made  in  the  skin,  and  the  material 
injected  with  a  syringe  having  a  dull  needle.  The  needle 
is  introduced  into  the  abdominal  cavity  in  the  median 
line;  into  the  pleural  cavity  at  the  upper  edge  of  a  rib. 

4.  Inoculation  into  the  Bloodvessels. 

In  rabbits  the  injection  is  made  into  one  of  the  large 


364     CHEMISTRY,  MICROSCOPY,  AND  BACTERIOLOGY 

veins  at  the  border  of  the  ear;  in  larger  animals  into  the 
jugular  vein. 

5.  Inoculation  into  the  Anterior  Chamber  of  the  Eye.— 

(Cf.  p.  20,) 

6.  Inoculation  by  Means  of  Food. 

The   material   to   be   examined   is   mixed    with   the 
food. 


THE   COPYRIGHTS   OF   THIS   BOOK,  IN  ALL  ENGLISH-SPEAKING 
COUNTRIES,    ARE   OWNED   BY   REBMAN   COMPANY,    NEW   YORK 


INDEX 


Acetic  acid  in  stomach,  62 
Acetone  in  urine,  163 

test  of,  204 
Actinomycosis,  28,  31 
Adventitious    constituents   of 

the  urine,  175 
Agar  medium,  346 
Agglutination,  272 
Albumin,  detection  of,  142,  180 

in  urine,  141 

precipitate  in  cold,  150 
Albuminosis,  148 
Almeris  test,  172 
Aloin  test  for  blood,  67 
Alveolar  epithelial  cells,  33 
Ammonia  test  in  urine,  203 
Amoeba  in  faeces,  97 
Amylum,  96 
Anaerobic  cultures,  359 
Anchylostoma  duodenale,  103 
Angina  Vincenti  (Plautii),  11 
Animal  test  for  tubercle  bacil- 
li in  urine,  242 

Animal  tests,  methods  of,  363 
Anthrax  bacillus,  128,  301 
Anthrax  carbuncle,  301 
Antigen,  syphilitic,  280 
Antipyrin  in  urine,  178 
Apparatus  for  collecting  mate- 
rial, 1 

Arsenic  in  urine,  176 
Ascaris  lumbricoides,  102 
Ascites  agar,  355 


Babes-Ernst's  bodies,  3 
Bacillus  fusiformis,  11 

pyocyaneus,  52 

Bacteriological  examination  in 

diseases  of  the  skin,  299 

of  conjunctival  secretions, 

19 

of  faeces,  104 
of  fluids  by  puncture,  291 
in  nasal  secretions,  17 
in    secretions    of    the 

mouth,  1 
in  sputum,  37 
in  urine,  237 
Bacteriolysine,  275 
Bacterium  coli  in  urine,  239 
Balantidium  coli,  98 
Barsiekow's  medium,  108,  351 
Baumgarten' s    method    of 
staining   lepra  bacilli, 
332 

Benzidin  test,  67,  82,  172 
Biliary  acids,  83 
Biliary  concretions,  90 
Biliary  pigments  in  faeces,  82 
pigments  in  gallstones,  90 
pigments  in  stomach,  66 
pigments  in  urine,  168 
Biological  properties  of  bac- 
teria, test  of,  361 
Biuretic  reaction,  149 
Boiling  test  for  albumin  in 
urine,  145 


365 


INDEX 


Blood,  examination  of,  250 
in    contents    of   stomach, 

66 

in  faeces,  82 
in  urine,  170 
pigment,  170 
serum  agar,  354 
serum  as  medium,  353 
specimens,   staining   of, 

256,  338 
Blood-agar,  352 
Blood-cells,     morphology    of, 

259 
Blood-corpuscles,  counting  of, 

253 

Boas  "faden"  (threads),  73 
Borax  methylene  blue,  329 
Bothriocephalus  latus,  100 
Bouillon  as  medium,  345 
Brandberg's  method  of  testing 

albumin,  180 
Bread  as  medium,  348 
Butyric  acid  in  stomach,  62 

Capsules   of   anthrax    bacilli, 

staining  of,  335 
Carbohydrates,  in  faeces,  indi- 
rect estimation  of,  86 
in  urine,  151 
Carbol  fuchsin,  329 
Casts  in  urine,  230 
Celloidin  for  imbedding,  340 
Cercomonas  intestinalis,  98 
Cerebro  spinal  fluid,  289 
Charcot-Leyden  crystals,    30, 

36 

Chloride  in  urine,  199 
Cholera  vibriones,  120 

test  of,  in  faeces,  123 
Cholesterme  in  gallstones,  91 
Chromogen  of  the  urine,  166 


Chrysophanic  acid,  177 
Coatings  of  mouth  and  phar- 
ynx, 1 

Collection  of  material,  1 
Congo-paper  test,  59 
Conjunctival  secretion,  19 
Conradi-Drigalski  medium, 

106,  348 

Copaiba  balsam  in  urine,  179 
Creatin,    test    for,    in   urine, 

132 

Culture  media,  344 
Culture  method,  356 
Curschmann' s   spirals,  27, 

29 

Cut  sections,  339 
Cylindroids  in  urine,  232 
Czaplewski's  method  of  count- 
ing   tubercle   bacilli   in 
sputum,  40 
of  staining  same,  331 

Dermato-mycosis,  305 
Dextrose  in  urine,  151 
Diacetic  acid,  163 
Diazo  reaction,  174 
Dimethylamidoazobenzol,  test 

with,  60 
Diphtheria  bacilli,  2-8 

in  conjunctival  secretions, 
19 

in  nasal  secretions,  17 

staining  of,  332 
Diplobacillus  of  Friedlaender, 
17,  52 

of  Morax  and  Axenfeld,  21 
Diplococcus  flavus,  16 
Dittrich's  plugs,  27,  30 
Donne's   test  for  ous,  135 
Ducrey's  bacillus,  304,  343 
Dysentery  bacillus,  117 


INDEX 


367 


Echinococcus  cysts,  288 

hooks,  31      . 

in  urine,  235 
Eggs  of  intestinal  parasites, 

99-103 

Elastic  fibres  in  sputum,  35 
Endos  fuchsin  agar,  104,  349 
Enteroliths,  92 
Epithelium  in  faeces,  97 

in  urine,  224 
Erythrasma,  316 
Essbach's  method  of  estimat- 
ing albumin,  181 
Exudates,  286 

Fasces,  examination  of,  74 

dry  matter,  84 
Fat,  in  faeces,  81,  85,  96 

in  urine,  134 

Fatty  acids,  volatile,  in  stom- 
ach, 

Favus,  309 
Fecal  balls,  83 
Fecal  concretions,  92 
Fecal  sieve,  76 
Fehling's  test,  156 
Fermentation   test    according 
to   Roberts,  183 

to  Lohnstein,  184 
Fibrin  in  urine,  151,  229 
Picker's  diagnosticum,  275 
Flagella,  staining  of,  336 
Formic   acid   in   gastric    con- 
tents, 62 
Freeing  the  urine  of  albumin, 

149 

Freezing-point  of  urine,   how 
to  determine,  137 

of  blood,  251 
Fruit  sugar,  160 
Fungi,  staining  of,  337 


Gabbet-FraenkeV s   method   of 

staining  tubercle  bacilli, 

331 

Gqffky's  scale,  41 
Gastric  contents,  examination 

of,  56,  68 

Gelatine  as  medium,  346 
Gerhardt's  test,  164 
Giemsa  stain,  258,  339 
Glucose  in  urine,  151 
Glycuronic  acid,  162 
Gmelin's  test,  83,  168 
Gonococci,  cultivation  of,  247 
in  conjunctival  secretions, 

20 

in  urine,  246 
Gonorrhoeal  threads,  234 
Gram's  staining  method,  330, 

342 

Grape-sugar  in  urine,  151 
Gravimetric  analysis,  182 
Green  solution,  Loeffler's,  112, 

349 
Guaiacum   test   after    Weber, 

66,  82 
Guensburg's  reaction  of  HC1, 

59 
Gutceit's  detection  of  arsenic, 

176 

Haemato-porphyrin,  173 
Haemoglobin  in  urine,  170 

test  in  blood  of,  251 
Haemolytic  system,  282 
Hasmosyderine,  34 
Hair,  examination  of,  306 
Hanging  drop,  327 
Heart-failure  cells,  34 
Heller's  test  of  albumin,  143 

of  blood,  171 
Hesse's  medium,  44,  352 


INDEX 


Hopkins'   method  of  estimat- 
ing uric  acid,  193 
Huppert's  test  for  biliary  pig- 
ment, 83,  169 

Hyaline  casts  in  urine,  231 
Hydrobilirubin  in  faeces,  83 
Hydrochloric  acid,  estimation 

of,  69 
free,  58 
total,  70 

Hydrogen-sulphide,  68 
Hydronephrosis,  288 

Indican  in  urine,  166 
Indol  reaction,  112 
Indoxyl  sulphuric  acid,  165 
Influenza  bacillus,  51 
Infusoria  in  faeces,  98 
Inoscopy,  293 
Intestinal  gravel,  93 

parasites,  97 
Iodine  test  in  urine,  177 

Jacobi's   method   of   determi- 
ning pepsin,  63 

Kernels  in  sputum,  26 
Kiefer's  ascites  agar,  355 
Kjeldahl's  method  of  estimat- 
ing nitrogen  in  faeces,  85 
in  urine,  187 
Koch-Week's  bacillus,  20 
Kowarsky's  copper  plate,  257 
method     of     determining 

uric  acid,  193 
pheryl-hydrozin   test,  158 
Kuehne-Weigert' s   method   of 
cut  staining,  338,  342 

Lactic  acid,  estimation  of,  72 
in  the  urine,  160 


Lactic  acid,  test  of,  60 
Lactose  in  urine,  160 
Lecithin  granules,  235 
Legal' s  test  of  acetone,  163 
Leischmann's  stain,  259 
Lepra  bacillus,  18 
Levaditi's  method,  322 
Levulose  in  urine,  160 
Lieben's  test  for  acetone,  164 
Liebermann-Allihn's    estima 

tion  of  starch  in  faeces, 

87 
Litmus-ascites-sugar  agar,  351 

litmus-whey,  108 
Liver  extract,  280 
Loeffler's  methylene  blue,  3 

serum,  4,  354 
Lohnstein's       saccharometer, 

184 
Ludwig-Salkow ski's      method 

of  estimating  uric  acid, 

197 

Malachite-green  agar,  107,  349 
Malaria  parasites,  263 
Mallei  bacilli,  300 
Hanson's  method  of  staining, 

264 

May-Gruenwald  stain,  258 
Meat-poisoning  bacillus,  117 
Melanin  in  urine,  174 
Meningococci,  13,  296 
Mercury  in  urine,  175 
Metahaemoglobin,  170 
Methyl  violet  as  reagent   for 

HC1,  60 
Melt's  method  of  determining 

pepsin,  63 

Micrococcus  catarrhalis,  15,  51 
cinereus,  16 
tetragenus,  50 
ureae,  136 


INDEX 


Microscopical  examination  of 
blood,  255 

of  faeces,  94 

of  gastric  contents,  72 

of  gonococci,  247 

of  urine,  209 
Microsporia,  312 
Microsporon  furfur,  315 
Milk  as  a  medium,  348 
Milk-sugar,  160 
Minz's   estimation   of   hydro- 
chloric acid,  69 
Mouth,  secretions  of  the,  1 

spirochetae  of  the,  12 
Mucin  in  faeces,  80 
Mucus  in  faeces,  78,  96 
Muscle  fibres  in  faeces,  95 

Nasal  secretions,  17 
Neisser's  stain,  3,  333 
Neutral  red  agar,  109,  351 
Nitrogen,  total,  estimation  of, 

85 

Nutrose  litmus  bouillon,  7 
Nylander's  test,  152 

Oidium  albicans,  10 
Onychomycosis,  314 
Oppler-Boas  threads,  73 
Ovarial  cysts,  314 
Oxalate     calcium     in     urine, 

214 
Oxalic  acid,  determination  of, 

202 

/2-Oxybutyric  acid,  163 
Oxyhsemoglobin,  170 
Oxyuris  vermicularis,  101 

Pancreatic  cysts,  289 
Pancreatic  stones,  93 


Pappenheim' s     method'  of 

staining  gonococci,  334 
tubercle  bacilli,  332 

Paraffin  embedding,  339 

Paratyphoid  bacillus,  115,  273 

Pavy-Sahli  method  of  estimat- 
ing sugar,  185 

Pentaglucose,  161 

Pentose,  161 

Pepsin,  62 

Pepsinogen,  62 

Peptone  solution,  347 

Peptones,  148 

Petruschky's  litmus-whey,  351 

Pfeiffer's  test,  275 

Pharynx,  1 

Phenacetin  in  urine,  178 

Phenol  in  urine,  179 

Phenyl-hydrazin  test,  158 

Phosphate  in   sediment  of 

urine,  216 
estimation  of,  199 

Pigment  of  urine,  165 

Pityriasis  versicolor,  315 

Plague  bacillus,  53,  128 

Plate  cultures,  356 

Plaut-Vincent' s  angina,  11 

Pneumo-bacillus,  52 

Pneumococci,  48 

Polar  granules,  3 

Polarization,  183 

Potassium   bromide  in   urine, 
177 

Potassium  iodide  in  urine,  177 

Potatoes  as  medium,  344 

Prostate,    secretions   of,   235, 
249 

Proteus  vulgaris,  245 
Hauseri,  136 

Pseudo-diphtheria  bacillus,  5 

Pseudo-mucin,  287 


370 


INDEX 


Pus  corpuscles  in  faeces,  97 
in  urine,  227 

Reaction  of  faeces,  80 

of  gastric  contents,  58 
of  urine,  135 
Red  blood-corpuscles,  counting 

of,  253 
in  faeces,  97 
in  urine,  229 
morphology  of,  259 
Relapsing  fever,    spirilla  of, 

268 

Renin,  65 
Reninogen,  65 

Roberts'  and  Stolnikojfs  meth- 
od, 180 
Roberts1     fermentation     test, 

183 

Rosenbach  modification,  169 
Rosin's  test,  169 
Roux's  solution,  3,  333 

Saccharometer  (Lohnstein's) , 

185 

Salicylic  acid  in  urine,  177 
Sarcinae,  72 

Schlesinger' s  method,  167 
"Schuettel"  cultures,  358 
Sedimentation,  42 
Sedimentum    lateritium,     134 
Serum  diagnosis,  272 
Skin  diseases,  299 
Smegma  bacillus,  241 
Soor  fungus,  10 
Spectroscopical   tests,  67,  82, 

172 

Spermatozoa,  235 
Spiegler's  test  of  albumin,  146 
Spiral  cells,  72 
Spirocheta  buccalis,  12 


Spirocheta  pallida,  317-326 

various  methods  of  stain- 
ing of,  320-324 
Spores,  staining  of,  334 
Sputum,  examination  of,  22 
Stab  cultures,  358 
Staining  methods,  329 

solutions,  329 
Staphylococ.ci  in  faeces,  127 

sputum,  50 

urine,  240 
Starch    in   faeces,    estimation 

of,  87 

Stomach,  see  Gastric  Contents 
Stomatitis  ulcerosa,  13 
Stones,  composed  of  drugs,  93 

light,  93 
Streptococci  in  faeces,  127 

in  sputum,  49 

in  urine,  240 
Stukowenkojf  s    detection     of 

mercury,  175 
Sugar,  estimation  of,  182 
Sulphates  in  urine,  201 
Sulphosalicylic  acid  test,  145 
Sykosis,  312 

Taenia,  cucumerina,  101 

flavopunctata,  101 

nana,  101 

saginata,  99 

solium,  99 

Test-breakfast  of  Ewald,  56 
Tetanus  bacillus,  302 
Toepfer's   estimation   of  free 

hydrochloric  acid,  69 
Transudates,  286 
Trichocephalus  dispar,  102 
Trichomonas  intestinalis,  98 
Trichophytosis,  311 
Trommer's  test,  154 


INDEX 


371 


Tubercle  bacilli  in  faeces,  126 

in  sputum,  40,  46 

in  urine,  240 

in   secretions  of   conjunc- 
tiva, 19 

in  secretions  of  nose,  17 

staining  of,  330,  343 
Typhoid  bacilli  in  blood,  269 

in  faeces,  104,  113 

in  urine,  244 

Urates,  212 
Urea,  131,  193 
Urethral  secretions,  245 
Uric  acid,  131 

in  sediment,  211 
Urinary  calculi,  206 
Urinary  concretions,  206 
Urine,  casts  of,  230 

collection  of,  129 

filaments  of,  234 


Urine,   general  properties  of, 

132,  141 

identification  of,  130 
sediment  of,    microscopic 

examination  of,  209 
specific  gravity  of,  136 
Urobilin,  167 
Urobilinogen,  167 
Urotropin  in  urine,  179 

Wassermann' s  reaction,  278 
Weber's    test   for   blood,    66, 

82 
Widal's  reaction,  274 

Xanthin  stones,  206,  208 
Xerosis  bacilli,  8 

Ziehl-Neelsen's  staining  of  tu- 
bercle bacilli,  330 
Ziehl's  solution,  2,  11,  329 


PLATE  I 


FIG.  A. -Diphtheria  Bacilli  from  a  Serum  Plate.  Stained 
according  to  Roux.  Magnification,  1:1,000  (After 
Czaplewski.)  Seepages. 


FIG.  B. -Diphtheria  Bacilli  from  a  Serum  Plate.  Stained 
according  to  Neisser.  Magnification.  1 ;  1,000.  (After 
Czaplewski. )  See  page  3. 


PLATE    II 


FIG.  C. — Smear  from  Sputum  containing  numerous  Tubercle 
Bacilli.  Stained  according  to  Ziehl-Neelsen.  After  Czap- 
lewski.)  See  page  40. 


PlG.  D. — Smear  from  Pneumonic  Sputum.     Stained  according 
to  Gram.     (After  Czaplewski. )    See  page  48. 


PLATE    III 


FIG.  E.— Smear  from  Pneumonic  Sputum.    Stained  with  Carbol- 
fuchsin.     (After  Czaplewski. )     See  page  48. 


FIG.  F.—  Smear  from  Bronchial  Sputum  in  a  Case  of  Catarrhal 
Bronchitis.  (Specimen  from  Professor  Kolle.)  Stained 
with  dilute  Carbol-fuchsin.  Magnification,  1 : 1,000.  (After 
Czaplewski. )  See  page  51. 


PLATE   IV 


FIG.  G.  —Smear  from  Pulmonary  Sputum  containing  Influenza 
Bacilli.  Stained  with  fuchsin.  (Specimen  from  Professor 
Kolle,  drawn  by  Landsberg,  Berlin. )  See  page  51. 


PLATE    V 


FIG.  H. — Smear  from  a  Cholera  Dejection.  A  Mucus  Fleck  con- 
taining an  almost  Pure  Culture  of  Comma  Bacilli.  The  Shoal 
arrangement.  Stained  with  Dilute  Carbol-fuchsin.  Mag- 
nification, 1 :  500.  (After  Kolle.)  See  page  120. 


FIG.  I.—  Uri<?  Acid.     See  page  211, 


PLATE    VI 


FIG.  J.  — Ammonium  Urate.     See  page  213. 


PLATE   VII 


FIG.   K. — Nephritis   in   Pregnancy.      Eclampsia.      Hematoidin 
Crystals.     (After  Blumenthal.)     See  page  222. 


PLATE    VIII 


FIG.  L. — a,  Casts  from  icteric  urine;  6,  Renal  epithelial  cells 
from  icteric  urine ;  c,  Leucocytes  from  icteric  urine.  See 
page  230. 


PLATE   IX 


FIG.  M.—  a,  Leucocytes  (pus-corpuscles)  ;  6,  Red  blood- 
corpuscles.     See  page  227. 


FIG.  N.— a,  Red  blood-corpuscles;  6,  Bundles  of  fibrin  fibres. 
See  page  229. 


PLATE    X 


FIG.  O.— a,  Hyaline  cast;  b,  Finely  granular  cast;  c,  Coarsely 
granular  cast ;  d,  Waxy  cast ;  e,  Red  blood-corpuscles.  See 
page  230. 


FIG.  P.— a,  Spermatozoa;   b,  Prostatic  corpuscles  (Corpora 
amylacea)  ;  c,  Spermin  crystals.     See  page  235. 


PLATE   XI 


FIG.  Q.— o,  Echinococcus  booklets ;  6,  Echinococcus  membrane ; 
c,  Red  blood-corpuscles ;  d,  Leucocytes.     See  page  235. 


FIG.  R. — Smear  from  Urinary  Sediment  in  Cystitis  due  to 
Bacterium  Coli.       (After  W.  Scholtz. )     See  page  239. 


PLATE    XII 


FIG.   S.— Tubercle  Bacilli  in  the  Urine  in  Tuberculosis  of  the 
Bladder.     (After  W.  Scholtz.)     See  page  240. 


FIG.  T.— Gonococci  in  Urethral  Secretion.      (After  W.  Scholtz.) 
See  page  247. 


PLATE    XIII 


FIG.  U. — Macrocytes,  Microcytes,  Poikilocytes,  Erythroblasts, 
Normal  Leucocytes.    Triacid.    (After  Grawitz. )    See  page  262. 


FIG.  V.— Blood  Smear  made  shortly  before  the  Onset  of  a  Ter- 
tian Chill.  Parasite  in  the  Process  of  Division.  Stained 
according  to  Manson.  (After  Kolle. )  See  page  265. 


PLATE    XIV 


FIG.  W.— Blood  Smear  taken  during  the  Height  of  Tertian 
Fever.  Large  Rings  and  Full-grown  Pigmented  Parasites. 
Stained  according  to  Romanowski.  (After  Kolle.)  See 
page  265. 


FIG.  X.— Blood  Smear  taken  from  a  Case  of  Chronic  Tropical 
Malaria.  Large  Rings,  Crescents.  Stained  according  to 
Romanowski.  (After  Kolle. )  See  page  267. 


PLATE   XV 


FIG.  Y.— Spirilla  of  Relapsing  Fever.     (After  v.  Jaksch. ) 
See  page  268. 


\ 


f*  X 


I'1  / 


FIG.  Z.— Anthrax  Bacilli  in  Rabbit's  Blood. 
(After  v.  Jaksch.)     See  page  301. 


PLATE   XVI 


V   /'  \      " 

.vSvfv.  *S.»N\ 


FIG.  a. —Tetanus  Bacilli  (pure  culture).     (After  v.  Jaksch.) 
See  page  302. 


14  DAY  USE 

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